Melanocortin 1 receptor ligands and methods of use

ABSTRACT

The subject invention pertains to a modified MC1R peptide ligand comprising a peptide that is a melanocortin 1 receptor (MC1R) ligand and a functionality or linker, such as a click functionality, for conjugation to a surface or agent. The modified MC1R peptide ligand can be coupled, e.g., via a click reaction with a complementary click functionality attached, to a moiety to form an MC1R-targeted agent. Drugs, contrast agents, polymers, particles, micelles, surfaces of larger structures, or other moieties can be targeted to the MC1R. The subject invention also pertains to a MC1R peptide ligand-micelle complex comprising a peptide that is a melanocortin 1 receptor ligand connected via a click reaction product to a micelle. The micelle is stable in vivo and can target melanoma tumor cells by association of the peptide ligand with the MC1R or the tumor and selectively provide a detectable and/or therapeutic agent (such as an imageable contrast agent and/or anti-cancer agent) selectively to the tumor cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.15/263,028, filed Sep. 12, 2016, which is a continuation of U.S.application Ser. No. 14/117,949, filed Nov. 15, 2013, now U.S. Pat. No.9,441,013, which is the National Stage of International ApplicationNumber PCT/US2012/038425, filed May 17, 2012, which claims the benefitof U.S. Provisional Application Ser. No. 61/487,239, filed May 17, 2011,U.S. Provisional Application Ser. No. 61/487,245, filed May 17, 2011,U.S. Provisional Application Ser. No. 61/531,357, filed Sep. 6, 2011,and U.S. Provisional Application Ser. No. 61/618,144, filed Mar. 30,2012, which are hereby incorporated by reference herein in theirentirety, including any figures, tables, nucleic acid sequences, aminoacid sequences, and drawings.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No. CA097360awarded by the National Institute of Health (NIH). The Government hascertain rights in the invention.

The Sequence Listing for this application is labeled “2OB9114.TXT” whichwas created on Jun. 24, 2019 and is 6 KB. The entire contents of thesequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The incidence of malignant melanoma is rising faster than that of anyother cancer in the United States; reportedly, melanoma diagnosesdoubled from 1986 to 2001 [1]. Melanoma progression is associated withaltered expression of cell surface proteins, including adhesion proteinsand receptors [2-7]. It has been estimated that over 80% of malignantmelanomas express high levels of the melanocyte stimulating hormone(αMSH) receptor, melanocortin 1 receptor (MC1R)[8]; thus, MC1R has beeninvestigated as a target for selective imaging and therapeutic agents.MC1R belongs to a family of five G protein-coupled receptors (MC1R-MC5R)known as the “melanocortins.” The melanocortins have been discovered ina wide range of tissues and organs throughout the body, ranging from thehair/skin (MC1R)[9] and kidneys (MC5R)[10] to the adrenal gland(MC2R)[11] and hypothalamus (MC3R/MC4R)[12, 13] and are known to play arole in skin pigmentation, hair coloration, obesity, metabolism,diabetes, sexual behavior, erectile dysfunction, stress response andmood.[9-16] Endogenously, the agonists for the melanocortins are the α-,β-, γ-melanocyte stimulating hormones (MSH) and adrenocorticotropichormone (ACTH, MC2R specific), all of which contain the same centralsequence of His-Phe-Arg-Trp (SEQ ID NO:1)[17]. This high degree ofpharmacophore homology makes it difficult to design a selective ligandwhich is highly specific for receptor subtype.

Due to its high expression on the surface of melanomas, MC1R has beeninvestigated as a target for selective imaging and therapeutic agents,and a number of selective ligands have been developed[18-20]. The mostwell known of these, [Nle⁴,D-Phe⁷]-α-MSH, has been investigatedextensively by Chen who showed that ⁹⁹mTc-CGCG labeled NDP-α-MSH boundto melanomas with very high avidity (6.5% ID/g)[21]. Unfortunately,NDP-α-MSH has also been shown to possess relatively strong nanomolarbinding affinities with MC3R, MC4R and MC5R as well[22-24]. Suchoff-target binding is highly undesirable given the presence of thesereceptors in sensitive organs such as the kidney and brain. Aco-injection of lysine has been reported to diminish off-target bindingin the kidneys[21, 25, 26], and presumably most agents will not be ableto cross the blood-brain barrier; nonetheless, the need for thedevelopment of highly specific and selective ligands against MC1R is oneof importance.

The development of ligands that can be attached to micelles and/orliposomes and designed to selectively target cancer cells relative tohealthy organs represents a major hurdle in current research. Many suchattempts fail either from (1) a loss of affinity resulting from theattachment of small peptides to large micelles or liposomes; (2) aninherent instability that results in collapse before entering thevicinity of the tumor; or (3) a nanoparticle size that is too large toescape the vasculature. In order to effectively design targetedparticles, each of these issues must be addressed.

BRIEF SUMMARY OF THE INVENTION

It has been estimated that over 80% of malignant melanomas express highlevels of the melanocyte stimulating hormone (αMSH) receptor,melanocortin 1 receptor (MC1R). Although there have been ligandsdesigned to attach to micelles and selectivity target cancer cellsrelative to healthy organs, many fail either from (1) a loss of affinityresulting from the attachment of small peptides to large micelles (2) aninherent instability of the system in vivo; or (3) a nanoparticle sizethat is too large. According to embodiments of the invention, a seriesof hMC1R ligands known to be selective against hMC1R are modified forattachment to a polymer, gel, or surface of a particle, micelle, orother structure. The most selective ligand was appended to a triblockpolymer micelle via click chemistry to a 100 nm polymer micellecomprises a targeted micelle with a nanomolar binding affinity to hMC1R.

One aspect of the invention concerns a modified melanocortin 1 receptor(MC1R) peptide ligand, comprising an MC1R peptide ligand coupled to aclick functionality at the C-terminus or N-terminus of the MC1R peptideligand. In some embodiments, the click functionality is an alkyne. Themodified MC1R peptide ligand may comprise, for example, the amino acidmotif His-Phe-Arg-Trp (HFRW) (SEQ ID NO:1) or His-DPhe-Arg-Trp (HfRW)(SEQ ID NO:2). In some embodiments, MC1R peptide ligand is selectedfrom:

(SEQ ID NO: 3) 4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂; (SEQ ID NO: 4)H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂; or  (SEQ ID NO: 5)H-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂. 

Throughout the various aspects of the invention described herein, inaddition to click reactions, it should be understood that other methodsfor conjugating the MC1R peptide ligand to micelles or to otherstructures may be utilized. The attachment of peptide moieties tohydrophilic polymers are known in the art, as multiple bio-conjugationapproaches exist. As described herein, a preferred approach utilizes thecopper catalyzed click chemistry reaction between an alkyne and an azideto form a 1,2,3-triazole linkage. In some embodiments, a metal-freeclick reaction can be utilized to form a triazole linkage between theazide and alkyne moieties (Jewett J C and C R Bertozzi, “Cu-free clickcycloaddition reactions in chemical biology,” Chem Soc Rev., 2010 April;39(4): 1272-1279, which is incorporated herein by reference in itsentirety). Other bio-conjugation approaches include, but are not limitedto, the reaction between an amine and aldehyde to form an imine linkage;the oxidation of two thiol moieties to form a disulfide; the thioleneclick reaction between an alkene and a thiol to form a thioether(Killops K L et al., “Robust, Efficient, and Orthogonal Synthesis ofDendrimers via Thiol-ene “Click Chemistry”, JACS, 2008, which isincorporated herein by reference in its entirety); reaction between anamine an ester to form an amide; the Michael addition conjugationbetween an amine and a maleimide to form a secondary amine linkage; andthe reaction between an amine and an activated ester to form an amide.

Another aspect of the invention is a method of preparing a MC1R peptideligand of the invention, comprising: providing an MC1R peptide ligand;and covalently bonding a moiety comprising a click functionality to theC-terminus or N-terminus of the MC1R peptide ligand, wherein a MC1Rpeptide ligand comprising a click functionality is formed.

In some embodiments, the C-terminus or N-terminus of the MC1R peptideligand comprises a lysine (Lys) residue or other nitrogen-bearing,thiol-bearing, or —OH bearing residue, and wherein the moiety comprisingthe click functionality is covalently bound to the residue. In someembodiments, the residue at the C-terminus or N-terminus comprises theLys residue. Preferably, the moiety having the click functionalitycomprises a terminal alkynyl acid of 5 to 12 carbons.

Another aspect of the invention concerns a melanocortin 1-targeted agentcomprising: a MC1R peptide ligand; and a moiety (a “payload”), whereinthe MC1R peptide ligand and the moiety are covalently linked by a clickreaction product (a reaction product of a first click functionality anda complementary second click functionality). The moiety is one to bedirected to MC1R expressing cells, such as a drug, contrast agent,polymer, gel, particle, surface, or any combination thereof.

Another aspect of the invention concerns a method of delivering a moietyto cells expressing the melanocortin 1 receptor (MC1R), comprisingadministering an MC1R-targeted agent of the invention to the cells invitro or in vivo. In in vivo embodiments, MC1R-targeted agent may beadministered to a human or non-human animal subject systemically, orlocally at the site of the cells. In some embodiments, the MC1R-targetedagent is administered to the subject intravascularly (e.g.,intravenously or intra-arterially). In some embodiments, the moiety(“payload”) carried by the MC1R-targeted agent is an anti-cancer agent(such as a chemotherapeutic agent). Preferably, the anti-cancer agent isone having efficacy against MC1R-expressing cells (melanoma cells). Insome embodiments, the moiety carried by the MC1R-targeted agent is acontrast agent, such as imaging contrast agents. Examples of imagingcontrast agents include, but are not limited to, near infraredfluorescent dyes (e.g., ICG derivatives); gold for CT contrast; Gd,Tc99m or ¹¹¹In chelate for MRI or SPECT imaging; Yttrium forradiotherapy, 18-F, 11-C, 18-O, Gallium 64, Copper-64 for PET imaging.Examples of anti-cancer agents include, but are not limited to,alkylating chemotherapy agents such as melphalan or ifosfamide; andother systemic melanoma chemotherapies such as Dacarbazine, paclitaxel,and vincristine.

In some embodiments, the subject is one diagnosed with melanoma. In someembodiments, the subject is one diagnosed with melanoma, and the moietyof the MC1R-targeted agent comprises an anti-cancer agent havingefficacy for the treatment of melanoma (e.g., an agent that killsmelanoma cells or inhibits the growth of melanoma cells). In someembodiments, the subject has not been diagnosed with melanoma.

In some embodiments, the moiety carried by the MC1R-targeted agentcomprises an imaging contrast agent, and the delivery method furthercomprises imaging the subject using an imaging modality (e.g., with animaging device) appropriate for the administered contrast agent, therebydetermining the localization of the contrast agent within the subject(for example, to determine the location of MC1R expressing cells).

Another aspect of the invention concerns a pharmaceutical compositioncomprising an MC1R targeted agent of the invention, and apharmaceutically acceptable carrier.

Another aspect of the invention concerns a modified melanocortin 1receptor (MC1R) peptide ligand-micelle complex, comprising: an MC1Rpeptide ligand; and a micelle comprising an inner core, outer core andhydrophilic shell, wherein the MC1R peptide ligand is linked to theshell of the micelle by a click reaction product. In some embodiments,the inner core of the micelle comprises a hydrophobic polypeptide, theouter core comprises a crosslinked peptide comprising a multiplicity ofcrosslinked amino acid residues and the hydrophilic shell comprises awater soluble polymer, and the inner core is covalently attached to theouter core and the outer core is covalently attached to the hydrophilicshell. In some embodiments, the water soluble polymer comprisespolyethylene glycol. In some embodiments, the click reaction productcomprises a 1,2,3-triazole from the addition of an azide and an alkyne.In some embodiments, the click reaction product is coupled to theC-terminus of the MC1R ligand. In some embodiments, the click reactionproduct is coupled to the N-terminus of the MC1R ligand. In someembodiments, the MC1R peptide ligand and a first click functionality ofthe click reaction product are selected from:

(SEQ ID NO: 3) 4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂;  (SEQ ID NO: 4)H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂;  or  (SEQ ID NO: 5)H-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂. In some embodiments, the MC1R peptide ligand comprises the amino acidmotif His-Phe-Arg-Trp (HFRW) (SEQ ID NO:1) or His-DPhe-Arg-Trp (HfRW)(SEQ ID NO:2).

In some embodiments, the MC1R peptide ligand-micelle complex furthercomprises an agent residing in the inner core of the micelle. The agentmay be an anti-cancer agent, contrast agent, or some other agent to beadministered.

Another aspect of the invention concerns a pharmaceutical compositioncomprising an MC1R peptide ligand-micelle complex, and apharmaceutically acceptable carrier.

Another aspect of the invention concerns a method of imaging a melanomatumor of a subject, comprising: administering the MC1R peptideligand-micelle complex of the invention to the subject, wherein acontrast agent is present within the inner core of the micelle, andwherein the MC1R peptide ligand-micelle complex concentrates in thetumor; and observing a signal from the contrast agent by an imagingdevice. In some embodiments, method of imaging comprises:

-   -   providing a MC1R peptide ligand-micelle complex of the        invention;    -   incorporating a contrast agent into the inner core of the MC1R        peptide ligand micelle complex;    -   administering the MC1R peptide ligand-micelle complex with the        contrast agent to a human or non-human animal subject, wherein        the MC1R peptide ligand-micelle complex concentrates in the        tumor; and    -   observing a signal from the contrast agent by an imaging device.

In some embodiments, the contrast agent comprises a near infrared (NIR)fluorescent dye, such as an ICG derivative. In some embodiments, thecontrast agent comprises a CT contrast agent, such as gold. In someembodiments, the contrast agent is a MRI or SPECT contrast agent, suchas Gd, Tc99m, or an ¹¹¹In chelate. In some embodiments, the contrastagent comprises a PET imaging agent, such as 18-F, 11-C, 18-O, orGallium 64.

Another aspect of the invention concerns a method of treating melanomatumor cells in a subject, comprising: administering the MC1R peptideligand-micelle complex of the invention to the subject, wherein ananti-cancer agent is present within the inner core of the micelle, andwherein the MC1R peptide ligand-micelle complex releases the anti-canceragent at the site of the tumor. Preferably, the anti-cancer agent is onethat demonstrates efficacy in the treatment of melanoma (for example,kills melanoma cells or inhibits the growth of melanoma cells). In someembodiments, the method comprises:

providing a MC1R peptide ligand-micelle complex of the invention;

-   -   incorporating an anti-cancer agent into the inner core of the        MC1R peptide ligand-micelle complex; and    -   administering the MC1R peptide ligand-micelle complex with the        anti-cancer agent to the subject, wherein the MC1R peptide        ligand-micelle complex releases the anti-cancer agent at the        site of the tumor.        In some embodiments, the anti-cancer agent is a radiotherapy        agent, such as Yttrium. In some embodiments, the anti-cancer        agent comprises an alkylating chemotherapy agent, such as        melphalan or ifosfamide. In some embodiments, the agent        comprises a systemic melanoma chemotherapy agent, such as        dacarbazine, paclitaxel, and/or vincristine.

Another aspect of the invention concerns a method of preparing a MC1Rpeptide ligand-micelle complex, comprising:

-   -   providing multiplicity of triblock polymer chains comprising a        hydrophobic polypeptide block attached to a central        crosslinkable peptide block comprising a multiplicity of        crosslinkable amino acid residues attached to a water soluble        polymer block, wherein a portion of the triblock polymer chains        further comprise a first click functionality covalently attached        to the water soluble polymer block distal to the central        crosslinkable peptide block and wherein the triblock polymer        chains self assemble into a micelle;    -   providing a MC1R peptide ligand comprising a complementary        second click functionality covalently attached to a MCIR        targeting peptide; and    -   combining the triblock polymer chains with the MC1R peptide        ligand, wherein the first click functionality and the        complementary second click functionality react to form a        reaction product that covalently joins the triblock polymer to        the MC1R peptide ligand to form a MC1R peptide ligand-micelle        complex.

In some embodiments, the MC1R peptide ligand is4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂ (SEQ ID NO:3);H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂(SEQ ID NO:4); orH-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂(SEQ ID NO:5).

In some embodiments, the method for preparing a MC1R peptideligand-micelle complex further comprises crosslinking the multiplicityof crosslinkable amino acid residues. In some embodiments, the methodfurther comprises the incorporation of an agent. In some embodiments,the agent comprises a contrast agent, anti-cancer agent, or both. Insome embodiments, the anti-cancer agent is a chemotherapeutic agent,such as a taxane.

Fluorescent and MRI targeted molecular imaging probes have been designedthat bind strongly and specifically to the melanocortin 1 receptor(MC1R) that is found expressed on the surface of over 80% of melanomacells. In some embodiments, the binding portion of the probes is theMC1R ligand with the structure 4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂ (SEQ ID NO:3) that shows high (0.2 nM) bindingaffinity for MC1R. Functionalization of the ligand at the C-terminuswith an alkyne for use in Cu-catalyzed click chemistry was used toattach a fluorescent dye or a micelle. Fluorescent targeted molecularimaging probes were developed by binding the MC1R ligand to a nearinfrared fluorescent dye, and cellular uptake of the probe byreceptor-mediated endocytosis was shown in engineered A375/MC1R cells invitro as well as in vivo by intravital fluorescence imaging with higheruptake values in tumors with high expression of MC1R compared to low(P<0.05). MRI targeted molecular imaging probes were developed bybinding the MC1R ligand to a micelle containing a gadolinium texaphyrin(Gd-Tx) chelate. These Gd-Tx micelles, stabilized by crosslinking withFe(III), are able to actively target MC1R expressing xenograft tumors invitro and in vivo suggesting that appropriately designed micelles mayeventually be able to deliver therapeutic payloads. The incidence ofmalignant melanoma is rising faster than that of any other cancer in theUnited States, with diagnoses having doubled from 1986 to 2001. Nodalmetastases are frequently the initial manifestation of metastatic spreadin patients with melanoma and accurate determination of nodal status isimportant for treatment planning. Currently, sentinel lymph nodebiopsies are performed to determine if the cancer has metastasized;however, one study showed only 24.3% (288/1184) of melanoma patients whohad a sentinel lymph node biopsy had metastases. The diagnostic probesof the invention can be used to determine if a biopsy is avoidable. Forexample, the probes can be administered to subjects with melanoma beforeand/or after treatment and the detectable signal from the probe isobserved to check nodal status and ascertain whether the cancer hasmetastasized. If a positive signal is detected, further diagnosticmethods may be utilized, such as sentinel nodal biopsy, and/or therapymay be undertaken.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A synthetic route that was used for compound 4, in which a) (i)Fmoc-AA-OH (3 eq), HOCt or HOBt (3 eq), and DIC (3 eq) in DMF/DCM (10mL/1 g of resin) for amino acid couplings; b) Piperidine/DMF (1:10, 2+20minutes); (ii) 4-phenylbutyric acid (6 eq), and DIC (3 eq) in DMF/DCM;b) (i) Pd(0)tetrakistriphenylphosphine (0.01 eq),N,N′-dimethylbarbituric acid (5 eq) in degassed DCM (2×30 minutes) (ii)5-hexynoic acid (5 eq) and DIC (3 eq) in DMF/DCM c) (i) TFA-scavengerscocktail (91% trifluoroacetic acid, 3% water, 3% thioanisole, 3%ethanedithiol); (ii) ether extraction; (iii) HLPC purification.

FIG. 2: A reaction scheme for preparation of the MC1R peptide ligandmicelle complex according to an embodiment of the invention (SEQ IDNO:15).

FIGS. 3A-B: Representative competitive binding assays for targeted anduntargeted micelles on HCT116/MC1R cells.

FIGS. 4A-C: Representative competitive binding assays for (FIG. 4A)4-targeted polymer; (FIG. 4B) untargeted micelles; and (FIG. 4C)4-targeted micelles against HCT116/hMC1R cells. X-axis concentrationsfor (FIG. 4A) and (FIG. 4C) were normalized to the targeting group.

FIGS. 5A-B: Plot of absorbance of a Gd-Tx agent in a (FIG. 5A)4-targeted (ML21-1 targeted) crosslinked (XL) micelle and a (FIG. 5B)4-targeted (ML21-1 targeted) uncrosslinked (UXL) micelle for in vitrobinding of the 4-targeted micelles at nanomolar concentrations.

FIG. 6: Grid of in vitro MR T1 weighted signals from Gd-Tx containing4-targeted (T) and 4-free (UT) micelles that are crosslinked (XL) orcrosslink free (UXL) for various loadings of Gd-Tx in the micelles andtwo different concentrations of the micelles.

FIG. 7: Grid of in vitro MR T1 weighted signals from Gd-Tx containing4-targeted (T) and 4-free (UT) micelles that are crosslinked (XL) orcrosslink free (UXL), where the columns are of the same order as FIG. 6,for various loadings of Gd-Tx in the micelles showing an optimal signalat loadings of Gd-Tx at a concentration of about 1.3% Gd-Tx in themicelle.

FIG. 8: Tabulated averaged data of quantified MR T1 weighted signalsfrom the data displayed in FIG. 7.

FIG. 9: In vivo MR T1 weighted images of mice that were injected withmicelles to produce a 12 mg/mL Gd-Tx concentration with targeted oruntagged micelles that are crosslinked or uncrosslinked.

FIG. 10: A closer view of the in vivo MR T1 weighted images of Mice ofFIG. 9 for micelles with the 4-targeted crosslinked micelles, where thetumor images are indicated.

FIG. 11: A reaction scheme for preparation of the MC1R peptide ligandmicelle complex according to an embodiment of the invention (agadolinium-texaphyrin (Gd-Tx) micelle formulation) (SEQ ID NOs:16-17).

FIGS. 12-15: Results of europium-time resolved fluorescence (Eu-TRF)binding assays.

FIG. 16: Micelle characterization data from twelve samples.

FIG. 17: In vitro SCOUT phantoms of Gd-Tx micelle with a multiple TRSEMS. The TR calculation sequence consisted of TR values of 20, 10.99,6.03, 3.31, 1.82, 1.00, 0.55, 0.30, 0.17, 0.09 and 0.05 s; TE=8.62 ms;data matrix=128×128, 4 averages, 2 dummy scans, FOV=80 mm×40 mm or 40mm×90 mm and the slice thickness=1-2 mm. T1 values were calculated usingthe vnmrj software (Agilent Life Sciences Technologies, Santa Clara,Calif.), and values were verified using MATLAB (Mathworks, Natick,Mass.).

FIGS. 18-21: In vivo MRI results.

FIG. 22: Coronal-90, T1-weighted contrast-enhanced SEM images throughthe center slice of subcutaneous MC1R-expressing tumors. Arrowsindicated the location of the tumors. Data matrix=128×128; FOV of 40 mm(read)×90 mm (phase); 15 one-mm thick slices were taken with a 0.5 mmgap between slices; TR=180 ms; TE=8.62 ms; and 8 averages for total scantime of about 3 minutes per SEMS image. Images were processed usingMATLAB (Mathworks, Matick, Mass.).

FIG. 23: A synthetic route for Cy5 and IR800CW ligands (Scheme Si). a.Fmoc/tBu synthesis continued as follows: i) Fmoc-aa-OH (3 eq), HOBt (3eq), DIEA (6 eq), and HBTU (3 eq) in DMF for amino acid couplings; ii)Piperidine/DMF (1:4) for Fmoc deprotection; b. Aloc deprotection: i)Pd(0)TPP₄ (0.2 eq), dimethylbarbituric acid (5.0 eq) in DCM (0.5 M) for30 min′ ii) Trt-OH (3 eq), DIEA (6 eq), and HBTU (3 eq) in DMF; c.TFA-scavengers cocktail (91% trifluoroacetic acid, 3% water, 3%triisopropylsilane, and 1,2-ethylenedithiol (3%) for 4 hrs; d. IR800CWmaleimide or Cy5 maleimide (1 eq) in DMF.

FIGS. 24A-C: DNA microarray expression profile of MC1R in melanoma,other skin cancers and normal tissues (FIG. 24A). Data are representedas mean±SD. Note the log₁₀ scale. DNA microarray of primary humanmelanocytes (white), melanoma cell lines with the NRAS mutation (gray)and melanoma cell lines with the BRAF mutation (black) (FIG. 24B).Representative IHC staining of MC1R in normal skin with a pathologyscore of 3 and different types of melanoma with pathology score of equalto or greater than 4 (FIG. 24C).

FIGS. 25A-C: ICC of MC1R expression on the surface of A375 parental andengineered cells (FIG. 25A). Confocal micrographs of cells incubatedwith the nuclear marker DAPI (blue), the plasma- and plasma-membranemarker, WGA (green) and MC1R antibody-Alexa 555 (red). To inhibitcellular uptake, cells were incubated with antibodies and dyes at 4° C.for 10 min. The merged image shows colocalization of MC1R (red) withmembrane marker (green) indicating accumulation of the receptor on thecell-surface (yellow). Representative saturation binding assays forA375/hMC1R cells (FIG. 25B). A representative competition binding plotof MC1RL-800 imaging probe to A375/hMC1R cells (FIG. 25C).

FIGS. 26A-B: Uptake studies of MC1R targeted imaging probes in vitro andin vivo. FIG. 26A shows uptake of the MC1RL-800 probe on A375/MC1Rcells. The probe is detected on the cell membrane as early as 15 secondsafter incubation and internalized into the cells by 5 min afterincubation. No binding or uptake of the probe was observed when highconcentrations of NDP-α-MSH was used as a blocking agent before addingthe probe to the cells. FIG. 26B shows in vivo uptake studies of theMC1R-635 probe using the dorsal skin-fold window chamber xenograft tumorfor intravital confocal imaging. The fluorescent signal from the probewas detected on the cell surface (yellow arrow) by 5 min after i.v.injection of the probe, and was detected inside the cells 24 h afterinjection.

FIGS. 27A-1, 27A-2, and 27B: In vivo and ex vivo targeting of MC1RL-800.FIG. 27A-1 shows a representative image of normalized fluorescenceintensity maps of a mouse bearing xenograft tumors, 2 hours postintravenous injection of the probe (left mouse): A375 cells thatconstitutively express low levels of MC1R were used to form thelow-expressing tumor (left flank) and A375/MC1R cells were used to formthe high expressing tumor (right flank). A blocking experiment wasperformed using co-injection of 0.25 μg unlabeled NDP-α-MSH and 5nmol/kg of the MC1RL-800 probe to determine target specificity (rightmouse). Inset (FIG. 27A-2) is showing quantification of MC1RL-800normalized fluorescent count of low and high expressing tumors incontrol and blocking animals. Data represent mean±s.d. NC: NormalizedCount (FIG. 27B) are ex vivo images of low- and high-expressing tumorswith corresponding IHC staining of MC1R.

FIGS. 28A, 28B-1, and 28B-2: FIG. 28A shows ex vivo bio-distribution ofMC1RL-800 in the tumors, kidneys and liver at different time-pointspost-injection. No signal was detected in the heart, lung, brain andother organs (not shown). The values were normalized as percentage ofthe highest signal. FIGS. 28B-1 and 28B-2 show mathematical modeling ofex vivo and in vivo bio-distribution of the probe, respectively. Sim:simulation, T: Tumor, K: kidneys.

FIGS. 29A-1, 29A-2, and 29B: Reduction of MC1RL-800 kidney uptake. FIGS.29A-1 and 29A-2 show pharmacokinetics of different concentrations ofinjected probe in MC1R low expressing and high expressing tumors andkidneys. Note that the lower concentration (1 nmol/kg) has the lowestkidney accumulation. Insets show representative images of normalizedfluorescence intensity maps of a mouse bearing xenograft tumors, 2 hourspost intravenous injection of the probe for each concentration. FIG. 29Bshows co-injection of 1 nmol/kg of MC4R/5R compound and 5 nmol/kg ofMC1RL-800 (right image) to reduce kidney uptake of the probe. Insetshows representative images of mice bearing xenograft tumors, 30 minpost intravenous injection of the probe (control, left image) and probeplus MC4R/5R compound (co-injection, right image). Data representmean±s.d. NC: Normalized Counts.

FIGS. 30A-D: Pharmacokinetics of different MC1RL-800 probeconcentrations injected into mice bearing low- and high-MC1R expressingtumors and kidneys (1 nMol/Kg, 5 nMol/Kg, 10 nMol/Kg, and 30 nMol/Kg,respectively). Note that the peak signal in the tumors and kidneysoccurs 2-hours post-injection and the lowest probe concentrationinjected had the lowest kidney accumulation. Data represent mean±s.d.

FIGS. 31A, 31B-1, and 31B-2: FIG. 31A shows verification of ratmicrovessel patency using Multiphoton Laser Scanning Microscope. Micewere intravenously injected with Blue Dextran to verify GFP ratmicrovessel (green) patency. Note the flowing of the Dextran in greenmicrovessels. FIGS. 31B-1 and 31B-2 show in vivo uptake studies of theMC1RL-800 probe using the dorsal skin-fold window chamber xenografttumor for intravital confocal imaging. The probe (red) has been takeninto the tumor surrounded by GFP microvessels (green) at 24 hr afterinjection.

FIGS. 32A-C: MC1RL-800 concentration and location combined with the 3Dprobe profile in the tumors using Optix-MX3 imaging system in vivo andex vivo. FIG. 32A shows 3D topography of a representative mouse bearingA375 xenograft tumor (left leg) and A375/MC1R tumor (right leg) 2 hourspost intravenous injection of the probe and ex vivo image of the sametumor. FIG. 32B shows A357/MC1R slices from top to bottom through thetumor volume. The distance from the surface of the scanning stageunderneath the mouse is indicated in mm underneath each slice. FIG. 32Cshows an enlarged image of the currently selected slice (green squaredlabeled image, 13.48 in the ex vivo panel of FIG. 32B). The color bar inFIG. 32C indicates the corresponding concentration of probe. Note thatthe ex vivo image and IHC staining of the center slice from the sametumor is shown in FIG. 27B.

FIG. 33: Dorsal skin-fold window-chamber xenograft tumor model in SCIDmouse. Intravital confocal imaging of MC1R-800CW probe tumor cell uptakein vivo using the dorsal skin-fold window chamber xenograft tumor modelis shown in FIG. 26B.

FIG. 34: Formulation of Gd-Tx micelles. Detailed information of theGd-Tx crystal structure can be found in the Supporting Information.

FIG. 35: Effect of % ligand 2 coverage on micelle binding avidity.

FIG. 36: Multiple TR SEMS (spin echo) images were acquired to calculatethe T1 values of a Gd-Tx micelle MR phantom prepared with samplescontaining Gd-Tx at 0.01 mg/mL with varying weight loading percentagesand micelle concentrations. T1 values from each row are presented as anaverage for simplicity. A table of the T1 values for each well isavailable in the Supplemental Information.

FIG. 37A-B: Coronal-90 T1 weighted spin echo multi slice (SEMS) imagesof mice treated with different Gd-Tx micelle formulations. A)Representative images from each group of mice treated with 0.5% Gd-Txmicelles at selected time points. White arrows denote location oftumors. B) Pre-injection and 24 h post-injection image of the 5% T-XLformulation loaded with 5% Gd-Tx (w/w).

FIG. 38A-D: Buildup and clearance data of Gd-Tx contrast enhancement in(a-b) tumor, (c) liver and (d) kidney. p-values are in comparison toT-XL group. All groups contained 3 mice except where noted. ^(⊥)Onemouse expired between 24 h and 48 h time point. ⁺One mouse expired uponinjection of micelle agent.

FIG. 39: Synthesis of Gd-Texaphyrin (Gd-Tx) 6.

FIG. 40A-C: Low resolution ESI-MS spectrum for 6.

FIG. 41: High resolution ESI-MS spectrum for 6.

FIG. 42A-D: HPLC spectrum for 6.

FIG. 43A-B: Xenograft tumor Expression of MC1R. In-vivo characterizationof MC1R surface expression for HCT116/MC1R cells. (a-b) IHC staining ofrepresentative left (a) and right (b) tumors from a SCID mouse. Bothwere scored as 3 for MC1R by a Board Certified Pathologist.

FIG. 44: Multiple TR SEMS (spin echo) images were acquired to calculatethe T1 values of a Gd-Tx micelle MR phantom prepared with samplescontaining Gd-Tx at 0.01 mg/mL with varying weight loading percentagesand micelle concentrations. Individual T1 values are located under eachwell.

FIG. 45: View of the Gd-Tx complex 6 after showing a partial atomlabeling scheme. Displacement ellipsoids are scaled to the 50%probability level. The hydrogen atoms were omitted for clarity.

FIG. 46: Side view of the Gd complex 6 showing a partial atom labelingscheme. Displacement ellipsoids are scaled to the 50% probability level.The hydrogen atoms were omitted for clarity.

DETAILED DISCLOSURE OF THE INVENTION

According to an embodiment of the invention, MC1R-ligands are selectedfrom those disclosed in the literature [22, 27, 28] for theirselectivity to MC1R over other G protein-coupled receptors and modifiedfor attachment to a surface, for example the surface of a polymer-basedmicelle. For example, one ligand that displays a high affinity for MC1R,with little-to-no interaction with MC4R and MC5R, is functionalizationand attached to a 100 nm micelle where attachment does not significantlyalter its binding affinity to MC1R. In one embodiment of the inventionthe attachment of the ligand comprises a functionality that can reactwith a complementary functionality on a polymer, such as a polymer-basedmicelle, via click chemistry. Some ligands with MC1R binding affinityare given in Table 1, below.

TABLE 1  Structures of ligands screened for MC1R selectivity. CompoundStructure 1 4-phenylbutyryl-His-DPhe-Arg-Trp-NH₂  (SEQ ID NO: 6) 2Ac-Homophenylalanine-His-DPhe-Arg-Trp-NH₂ (SEQ ID NO: 7) 34-hydroxycinnamoyl-His-DPhe-Arg-Trp-NH₂  (SEQ ID NO: 8) 44-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂ (SEQ ID NO: 3) 5H-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂ (SEQ ID NO: 9) 6H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂  (SEQ ID NO: 4) 7H-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂ (SEQ ID NO: 10) 8H-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂  (SEQ ID NO: 5)

MC1R-selective ligands evaluated include: one based on LK-184 (compound1); [20, 29] compound 2, an N-terminal amino analog compound tofacilitate attachment to nanoparticles structures; compound 3 where theflexible —(CH₂)₃— of compound 1 is replace with a more rigid double bond(—CH═CH—) and with a 4-hydroxy function incorporated with the phenylring as an alternative N-terminal attachment point; and peptides knownfrom a prior study (compounds 5 and 7) [22]. The 4-phenylbutyroyl ofcompound 1 was substituted with an Ac-homophenylalaninyl (HPE) moiety ofcompound 2, wherein the amino group is an alternative attachment pointto the C-terminal attachment via a lysine ξ-amino as in compound 4.Compounds 4, 6, and 8, are ligands comprising a click functionality, analkyne, for attachment to a moiety comprising a complementaryfunctionality.

In general, unless otherwise specified, the abbreviations used for thedesignation of amino acids and the protective groups used therefore arebased on recommendations of the IUPAC-IUB Commission of BiochemicalNomenclature (Biochemistry, 11:1726-1732 (1972)). The nomenclature usedto define compounds of the invention is that specified by IUPAC,published in European Journal of Biochemistry, 138:9-37 (1984). Withregard to certain amino acids disclosed herein, their structures andabbreviations are apparent from the peptide structure below.

The letter “D” preceding any three-letter abbreviation for an aminoacid, e.g. as in “D-Nal” or “D-Phe,” denotes the D-form of the aminoacid. The letter “L” preceding an amino acid three-letter abbreviationdenotes the natural L-form of the amino acid. For purposes of thisdisclosure, unless otherwise indicated, absence of a “D” or “L”designation indicates that the abbreviation refers to both the D- andL-forms. Where the common single-letter abbreviation is used,capitalization refers to the L-form and small letter designation refersto the D-form, unless otherwise indicated. For each amino acid, anadditional conservative substitution includes the “homolog” of thatamino acid, where the “homolog” is an amino acid with a methylene group(CH₂) inserted into the side chain at the beta position of that sidechain. Examples of such homologs include, without limitation,homophenylalanine, homoarginine, homoserine, and the like. As usedherein, the term “peptide,” is defined as an amino acid sequence fromthree amino acids to about 700 amino acids in length.

As used herein, “MC1R ligand” and “ligand” refer to a compound withaffinity for melanocortin receptors, particularly melanocortin 1receptors, that can result in measurable biological activity in cells,tissues, or organisms that contain the MC receptor or blocks stimulationby a known MC agonist. Assays that demonstrate melanocortin receptoragonistic or antagonist activity of compounds are well known in the art.

Related peptides includes allelic variants; fragments; derivatives;substitution, deletion, and insertion variants; fusion polypeptides; andorthologs; and each amino acid of each such related peptide may beeither natural or unnatural of the “D” or “L” configuration whichcorresponds to the stereochemical designation “S” and “R,” respectively,as defined in the RS system of Cahn et al., (Pure Applied Chemistry,45:11-30 (1974), and references cited therein). Such related peptidesmay be mature peptides, i.e., lacking a signal peptide.

Embodiments of the invention are directed to a MC1R-ligand comprising areactive functionality towards a complementary functionality on a moietysuch as a small molecule, a polymer or a functionalized surface, forexample a functionality on the polymer shell of an in vivo stablemicelle wherein the MC1R comprises a functionality that allowsattachment via a Huisgen 1,3-dipolar cycloaddition, Diels-Alderreaction, or any other reaction that has the features of “click”chemistry to a complementary functionality on the moiety. Clickchemistry involves a reaction that displays selectivity and highconversion, generally although not necessarily, without driving thereaction by removal of a side product. In addition to use with a polymershell of a micelle, the MC1R-ligand can be attached as end groups orside groups of a water soluble or water suspendable homopolymer orcopolymer. The homopolymer or copolymer can be linear, branched,hyperbranched, dendritic, or a network. The copolymer can be a randomcopolymer, block copolymer, or graft copolymer. Surfaces can be that ofa particle, including polymeric, ceramic, glass, or metal where thesurface is flat or irregular including within the pores of a solidporous material. The dimensions of particles can be in the nanometer,micrometer or of larger dimensions.

Some embodiments of the invention provide a stabilized, targeted micellesystem capable of delivering systemic diagnostic agents and/ortherapeutic agents (“theragnostics”) specifically to a tumor site.Gadolinium-texaphyrin (Gd-Tx) has been investigated as a radiationsensitizing agent, and is used for exemplification herein. In someembodiments, the therapeutic agent is a chemotherapeutic agent, such asa taxane.

The MC1R-ligand can be coupled via a linking group to a small molecule,polymer or functionalized surface that includes a contrast agent (e.g.,imaging contrast agent) or a therapeutic agent by a stable orbiodegradable linker. The contrast agents can include: near infrared(NIR) fluorescent dyes, such as ICG derivatives; CT contrast agents,such as gold; MRI or SPECT contrast agents, such as Gd, Tc99m, and ¹¹¹Inchelates; radiotherapy agents, such as Yttrium; PET imaging agentscomprising, for example, 18-F, 11-C, 18-O, or Gallium 64; alkylatingchemotherapy agents, such as melphalan or ifosfamide; and compounds forsystemic melanoma chemotherapies, such as dacarbazine, paclitaxel, andvincristine.

In some embodiments of the invention, the MC1R-ligand is attached to astable micelle (an MC1R peptide ligand-micelle complex) comprising adiblock, triblock or tetrablock copolymer that self organizes into: aninner core comprising a hydrophobic block that provides an environmentwhere a drug or other agent can reside within the micelle; an outer corecomprising a intermediate unit or block comprising at least one groupthat crosslinks, hence stabilizing the micelle; and a hydrophilic shellcomprising a water soluble polymer with a functionality distal to thecore wherein the functionality is used to attach the targetingMC1R-ligand. Micelles of this type are disclosed in Breitenkamp, et al.,U.S. Pat. No. 7,638,558, and incorporated herein by reference. Thecrosslink of the outer shell can be a chemical crosslink which compriseone or more covalent bonds or a physical crosslink that involveassociated functional groups or ions, which bind by ligation of ions,dipolar interactions, or any other intermolecular forces. The crosslinkcan be a disulfide, ester, hydrazone, Schiff base, zinc complexation,Iron (III) complexation, or other crosslinking that can be reversible.In embodiments of the invention, the crosslink is stable in vivo at anormal pH exhibited in most normal cells but uncrosslinks at the lowerpH of a malignant cell that is targeted, permitting delivery of apayload to a desired anatomical site, such as a tumor site, through apH-triggered mechanism. Barkey N M et al., “Development of MelanomaTargeted Polymer Micelles by Conjugation of a Melanocortin 1 Receptor(MC1R) Specific Ligand,” J. Med. Chem., October 2011, 54:8078-8084,which describes the formation of embodiments of MC1R peptideligand-micelle complexes of the invention, is incorporated herein byreference in its entirety (see, for example, compound #4 in Table 1 ofBarkey N M et al.).

Water soluble, hydrophilic, polymers that can be used includepolyethyleneoxide (also referred to as polyethylene glycol or PEG),poly(N-vinyl-2-pyrolidone), poly(N-isopropylacrylamide),poly(hydroxyethyl acrylate), poly(hydroxylethyl methacrylate),poly(N-(2-hydroxypropoyl)methacrylamide) (HMPA), or any derivativesthereof. Such water soluble polymers are prepared in a manner such thatthe distal end to the core has a reactive functionalized that iscomplementary to a reactive functionality on the targeting MC1R-ligand.In an embodiment of the invention, the reactive functionality on thehydrophilic polymer can be a triazine and the complementaryfunctionality on the MC1R-ligand can be a terminal alkyne. In anotherembodiment of the invention the reactive functionality on thehydrophilic polymer can be a terminal alkyne and the complementaryfunctionality on the MC1R-ligand can be a triazine.

In an embodiment of the invention, the micelle has an inner core thatcomprises a poly(amino acid) block where a sufficient proportion of theamino acid repeating units have a hydrophobic side group to render theblock hydrophobic. The amino acids can be natural or unnatural. Theamino acids can include phenylalanine, alanine benzyl glutamate, alkylglutamate, benzyl aspartate, alkyl aspartate, leucine, tyrosine, serine,threonine, glutamic acid, aspartic acid, or a combination thereof.

In some embodiments of the invention, the micelle has an outer corecomprising a reaction functionality that can be combined with a likereaction functionality to form a crosslink. For example, a pair of thiolfunctionality can be combined to form a disulfide. The combinedfunctionality can be with the inclusion of a disubstituted couplingreagent. For example, a carboxylic acid functionality can be combinedwith a divalent or polyvalent salt to form an ionic crosslink, orcondensed with a diol, diamine, or other symmetrically or asymmetricallydisubstituted reagent to form a covalent crosslink.

Some embodiments of the invention are directed to a method for thepreparation of the MC1R-ligands comprising a functional group that canundergo a click reaction, as shown in FIG. 1. The method involvespreparation of a peptide sequence comprising a 4-propynyl amide ateither the C terminal or, alternately, the N terminal end of thepeptide. The peptide can be prepared using a Rink Amide Tentagel resin(0.23 mmol/g) using a Fmoc/tBu synthetic strategy and standardactivations. The protected peptide is selectively deprotected withcleavage of the Aloc group, and subsequently condensed with a clickreagent containing compound, for example a 5-hexynoic acid, to form the5-hexynyl amide group, as the site for surface attachment of theMC1R-ligand. The remainder of the protection groups and the cleavagefrom the resin can be carried out by addition of a TFA scavengercocktail.

Some embodiments of the invention are directed to the functionalizationof a surface with a MC1R-ligand comprising a functional group for aclick reaction and a surface comprising the complementary functionality.In an embodiment of the invention, an MC1R-ligand comprises an alkynefunctionality at an amino acid residue at the C terminal end, oralternately at the N terminal end, is added to an azide functionalizedsurface of an in vivo stable micelle. In cases in which the surface isthe surface of a micelle, a MC1R peptide ligand micelle complex can beformed as shown in FIG. 2.

Some embodiments of the invention are directed to the delivery of drugs,contrast agents, or other agents attached to the MC1R-ligand orcontained within a particle or micelle that is attached to theMC1R-ligand, to a patient. The micelle can be an in vivo stable micellethat can de-crosslink at a low pH. The terms “subject”, “individual”, or“patient” as used herein refer to any human or non-human animal,including mammals, to whom treatment with a composition according to thepresent invention is provided. Mammalian species that benefit from thedisclosed methods of treatment include, and are not limited to, apes,chimpanzees, orangutans, humans, monkeys; domesticated animals (e.g.,pets) such as dogs, cats, guinea pigs, hamsters, rabbits, rats, mice,and ferrets; and domesticated farm animals such as cows, horses, swine,and sheep.

The methods of the present invention can be advantageously combined withat least one additional diagnostic and/or treatment method, includingbut not limited to, chemotherapy, radiation therapy, surgery,immunotherapy or any other therapy known to those of skill in the artfor the treatment and management of a cancer.

While MC1R peptide ligands of the invention can be administered to cellsin vitro and in vivo as isolated agents, it is preferred to administerthese MC1R peptide ligands as part of a pharmaceutical composition. Thesubject invention thus further provides compositions comprising apeptide of the invention in association with at least onepharmaceutically acceptable carrier. The pharmaceutical composition canbe adapted for various routes of administration, such as enteral,parenteral, intravenous, intramuscular, topical, subcutaneous,intratumoral, and so forth. Administration can be continuous or atdistinct intervals, as can be determined by a person of ordinary skillin the art.

The MC1R peptide ligands of the invention can be formulated according toknown methods for preparing pharmaceutically useful compositions.Formulations are described in a number of sources which are well knownand readily available to those skilled in the art. For example,Remington's Pharmaceutical Science (Martin, E. W., 1995, Easton Pa.,Mack Publishing Company, 19^(th) ed.) describes formulations which canbe used in connection with the subject invention. Formulations suitablefor administration include, for example, aqueous sterile injectionsolutions, which may contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient; and aqueous and nonaqueous sterile suspensions whichmay include suspending agents and thickening agents. The formulationsmay be presented in unit-dose or multi-dose containers, for examplesealed ampoules and vials, and may be stored in a freeze dried(lyophilized) condition requiring only the condition of the sterileliquid carrier, for example, water for injections, prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powder, granules, tablets, etc. It should be understood that inaddition to the ingredients particularly mentioned above, thecompositions of the subject invention can include other agentsconventional in the art having regard to the type of formulation inquestion.

Examples of pharmaceutically acceptable salts are organic acid additionsalts formed with acids that form a physiological acceptable anion, forexample, tosylate, methanesulfonate, acetate, citrate, malonate,tartarate, succinate, benzoate, ascorbate, alpha-ketoglutarate, andalpha-glycerophosphate. Suitable inorganic salts may also be formed,including hydrochloride, sulfate, nitrate, bicarbonate, and carbonatesalts.

Pharmaceutically acceptable salts of compounds may be obtained usingstandard procedures well known in the art, for example, by reacting asufficiently basic compound such as an amine with a suitable acidaffording a physiologically acceptable anion. Alkali metal (for example,sodium, potassium or lithium) or alkaline earth metal (for examplecalcium) salts of carboxylic acids can also be made.

The active agent (MC1R peptide ligands/complexes) may also beadministered intravenously or intraperitoneally by infusion orinjection. Solutions of the active agent can be prepared in water,optionally mixed with a nontoxic surfactant. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, triacetin, andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations can contain a preservative to prevent the growthof microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the compounds of the invention which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. The ultimatedosage form should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of therequired particle size in the case of dispersions or by the use ofsurfactants. Optionally, the prevention of the action of microorganismscan be brought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars, buffers or sodium chloride. Prolongedabsorption of the injectable compositions can be brought about by theinclusion of agents that delay absorption, for example, aluminummonostearate and gelatin.

MC1R peptide ligands/complexes of the invention may be administeredlocally at the desired anatomical site, such as a tumor site, or remotefrom the desired state, or systemically. Sterile injectable solutionsare prepared by incorporating the MC1R peptide ligands of the inventionin the required amount in the appropriate solvent with various otheringredients enumerated above, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying techniques, which yield a powder ofthe active ingredient plus any additional desired ingredient present inthe previously sterile-filtered solutions.

For topical administration, the agents may be applied in pure-form,i.e., when they are liquids. However, it will generally be desirable toadminister them topically to the skin as compositions, in combinationwith a dermatologically acceptable carrier, which may be a solid or aliquid.

The agents of the subject invention can be applied topically to asubject's skin to reduce the size (and may include complete removal) ofmalignant or benign growths. The MC1R peptide ligands of the inventioncan be applied directly to the growth. For example, the MC1R peptideligand may be applied to the growth in a formulation such as anointment, cream, lotion, solution, tincture, or the like. Drug deliverysystems for delivery of pharmacological substances to dermal lesions canalso be used, such as that described in U.S. Pat. No. 5,167,649 (Zook).

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the peptide can be dissolved or dispersed at effectivelevels, optionally with the aid of non-toxic surfactants. Adjuvants suchas fragrances and additional antimicrobial agents can be added tooptimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers, for example.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user. Examples of useful dermatological compositionswhich can be used to deliver the peptides to the skin are disclosed inJacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Woltzman (U.S.Pat. No. 4,820,508).

Useful dosages of the pharmaceutical compositions of the presentinvention can be determined by comparing their in vitro activity, and invivo activity in animal models. Methods for the extrapolation ofeffective dosages in mice, and other animals, to humans are known in theart; for example, see U.S. Pat. No. 4,938,949.

Patients in need of treatment and/or diagnosis using the compositionsand methods of the present invention can be identified using standardtechniques known to those in the medical or veterinary professions, asappropriate.

As used herein, the terms “cancer” and “malignancy” are usedinclusively. As used herein, the terms “cancer” and “malignancy” referto or describe the physiological condition in mammals that is typicallycharacterized by unregulated cell growth. The cancer may bedrug-resistant or drug-sensitive. Examples of cancer include but are notlimited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Moreparticular examples of such cancers include melanoma breast cancer,prostate cancer, colon cancer, squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, pancreaticcancer, cervical cancer, ovarian cancer, peritoneal cancer, livercancer, e.g., hepatic carcinoma, bladder cancer, colorectal cancer,endometrial carcinoma, kidney cancer, and thyroid cancer. The cancer maybe primary or metastatic. In some embodiments, the cancer is metastaticmelanoma.

Other non-limiting examples of cancers are basal cell carcinoma, biliarytract cancer; bone cancer; brain and CNS cancer; choriocarcinoma;connective tissue cancer; esophageal cancer; eye cancer; cancer of thehead and neck; gastric cancer; intra-epithelial neoplasm; larynx cancer;lymphoma including Hodgkin's and Non-Hodgkin's lymphoma; myeloma;neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, andpharynx); retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; sarcoma; skin cancer; stomach cancer; testicularcancer; uterine cancer; cancer of the urinary system, as well as othercarcinomas and sarcomas. Examples of cancer are listed in Table 15below.

As used herein, the terms “administering” or “administer” is defined asthe introduction of a substance (MC1R peptide ligand or complex) intocells in vitro or into the body of an individual in vivo and includesoral, nasal, ocular, rectal, vaginal and parenteral routes. The MC1Rpeptide ligand or complex may be administered individually or incombination with other agents via any route of administration, includingbut not limited to subcutaneous (SQ), intramuscular (IM), intravenous(IV), intraperitoneal (IP), intradermal (ID), via the nasal, ocular ororal mucosa (IN), or orally. For example, the MC1R peptide ligand orcomplex can be administered by direct injection into a tumor or at asite remote from the tumor.

The MC1R peptide ligand or complex can be administered to treat adisorder, such as cancer. As used herein, the terms “treat” or“treatment” refer to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent or slow down(lessen) an undesired physiological change or disorder, such as thedevelopment or spread of cancer or other proliferation disorder. Forpurposes of this invention, beneficial or desired clinical resultsinclude, but are not limited to, alleviation of symptoms, diminishmentof extent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. For example, treatment with a peptide of theinvention may include reduction of undesirable cell proliferation,and/or induction of apoptosis and cytotoxicity. “Treatment” can alsomean prolonging survival as compared to expected survival if notreceiving treatment. Those in need of treatment include those alreadywith the condition or disorder as well as those prone to have thecondition or disorder or those in which the condition or disorder is tobe prevented or onset delayed. Optionally, the patient may be identified(e.g., diagnosed) as one suffering from the disease or condition (e.g.,cancer) prior to administration of the peptide of the invention.

As used herein, the term “(therapeutically) effective amount” refers toan amount of the MC1R peptide ligand or complex of the invention orother agent (e.g., a drug) effective to treat a disease or disorder in amammal. In the case of cancer or other proliferation disorder, thetherapeutically effective amount of the agent may reduce (i.e., slow tosome extent and preferably stop) unwanted cellular proliferation; reducethe number of cancer cells; reduce the tumor size; inhibit (i.e., slowto some extent and preferably stop) cancer cell infiltration intoperipheral organs; inhibit (i.e., slow to some extent and preferablystop) tumor metastasis; inhibit, to some extent, tumor growth; and/orrelieve, to some extent, one or more of the symptoms associated with thecancer. To the extent the administered MC1R peptide ligand or complexprevents growth of and/or kills existing cancer cells, it may becytostatic and/or cytotoxic. For cancer therapy, efficacy can, forexample, be measured by assessing the time to disease progression (TTP)and/or determining the response rate (RR).

As used herein, the term “growth inhibitory amount” of the MC1R peptideligand or complex of the invention refers to an amount which inhibitsgrowth or proliferation of a target cell, such as a tumor cell, eitherin vitro or in vivo, irrespective of the mechanism by which cell growthis inhibited (e.g., by cytostatic properties, cytotoxic properties,etc.). In a preferred embodiment, the growth inhibitory amount inhibits(i.e., slows to some extent and preferably stops) proliferation orgrowth of the target cell in vivo or in cell culture by greater thanabout 20%, preferably greater than about 50%, most preferably greaterthan about 75% (e.g., from about 75% to about 100%).

The terms “cell” and “cells” are used interchangeably herein and areintended to include either a single cell or a plurality of cells, invitro or in vivo, unless otherwise specified.

In some embodiments, an agent is coupled to the MC1R peptide ligand orincorporated within the micelle of the complex. In some embodiments, theagent is an anti-cancer agent, such as a chemotherapeutic agent,biologic, etc. having anti-cancer activity.

As used herein, the term “anti-cancer agent” refers to a substance ortreatment (e.g., radiation therapy) that inhibits the function of cancercells, inhibits their formation, and/or causes their destruction invitro or in vivo. Examples include, but are not limited to, cytotoxicagents (e.g., 5-fluorouracil, TAXOL), chemotherapeutic agents, andanti-signaling agents (e.g., the PI3K inhibitor LY). In addition tobeing coupled to the MC1R peptide ligand or incorporated within themicelle, additional anti-cancer agents may be administered before,during, after administration of the MC1R peptide ligand or complex.Anti-cancer agents include but are not limited to the chemotherapeuticagents listed Table 14.

As used herein, the term “cytotoxic agent” refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells in vitro and/or in vivo. The term is intended to includeradioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², and radioactive isotopes of Lu), chemotherapeutic agents,toxins such as small molecule toxins or enzymatically active toxins ofbacterial, fungal, plant or animal origin, and antibodies, includingfragments and/or variants thereof.

As used herein, the term “chemotherapeutic agent” is a chemical compounduseful in the treatment of cancer, such as, for example, taxanes, e.g.,paclitaxel (TAXOL, BRISTOL-MYERS SQUIBB Oncology, Princeton, N.J.) anddoxetaxel (TAXOTERE, Rhone-Poulenc Rorer, Antony, France), chlorambucil,vincristine, vinblastine, anti-estrogens including for exampletamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andtoremifene (FARESTON, GTx, Memphis, Tenn.), and anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin, etc.Examples of chemotherapeutic agents that may be used in conjunction withthe compounds of the invention are listed in Table 14. In someembodiments, the chemotherapeutic agent is one or more anthracyclines.Anthracyclines are a family of chemotherapy drugs that are alsoantibiotics. The anthracyclines act to prevent cell division bydisrupting the structure of the DNA and terminate its function by: (1)intercalating into the base pairs in the DNA minor grooves; and (2)causing free radical damage of the ribose in the DNA. The anthracyclinesare frequently used in leukemia therapy. Examples of anthracyclinesinclude daunorubicin (CERUBIDINE), doxorubicin (ADRIAMYCIN, RUBEX),epirubicin (ELLENCE, PHARMORUBICIN), and idarubicin (IDAMYCIN).

TABLE 14 Examples of Chemotherapeutic Agents 13-cis-Retinoic AcidMylocel 2-Amino-6- Letrozole Mercaptopurine Neosar 2-CdA Neulasta2-Chlorodeoxyadenosine Neumega 5-fluorouracil Neupogen 5-FU Nilandron6-TG Nilutamide 6-Thioguanine Nitrogen Mustard 6-Mercaptopurine Novaldex6-MP Novantrone Accutane Octreotide Actinomycin-D Octreotide acetateAdriamycin Oncospar Adrucil Oncovin Agrylin Ontak Ala-Cort OnxalAldesleukin Oprevelkin Alemtuzumab Orapred Alitretinoin OrasoneAlkaban-AQ Oxaliplatin Alkeran Paclitaxel All-transretinoic acidPamidronate Alpha interferon Panretin Altretamine ParaplatinAmethopterin Pediapred Amifostine PEG Interferon AminoglutethimidePegaspargase Anagrelide Pegfilgrastim Anandron PEG-INTRON AnastrozolePEG-L-asparaginase Arabinosylcytosine Phenylalanine Mustard Ara-CPlatinol Aranesp Platinol-AQ Aredia Prednisolone Arimidex PrednisoneAromasin Prelone Arsenic trioxide Procarbazine Asparaginase PROCRIT ATRAProleukin Avastin Prolifeprospan 20 with Carmustine implant BCGPurinethol BCNU Raloxifene Bevacizumab Rheumatrex Bexarotene RituxanBicalutamide Rituximab BiCNU Roveron-A (interferon alfa-2a) BlenoxaneRubex Bleomycin Rubidomycin hydrochloride Bortezomib SandostatinBusulfan Sandostatin LAR Busulfex Sargramostim C225 Solu-Cortef CalciumLeucovorin Solu-Medrol Campath STI-571 Camptosar StreptozocinCamptothecin-11 Tamoxifen Capecitabine Targretin Carac Taxol CarboplatinTaxotere Carmustine Temodar Carmustine wafer Temozolomide CasodexTeniposide CCNU TESPA CDDP Thalidomide CeeNU Thalomid CerubidineTheraCys cetuximab Thioguanine Chlorambucil Thioguanine TabloidCisplatin Thiophosphoamide Citrovorum Factor Thioplex CladribineThiotepa Cortisone TICE Cosmegen Toposar CPT-11 TopotecanCyclophosphamide Toremifene Cytadren Trastuzumab Cytarabine TretinoinCytarabine liposomal Trexall Cytosar-U Trisenox Cytoxan TSPA DacarbazineVCR Dactinomycin Velban Darbepoetin alfa Velcade Daunomycin VePesidDaunorubicin Vesanoid Daunorubicin Viadur hydrochloride VinblastineDaunorubicin liposomal Vinblastine Sulfate DaunoXome Vincasar PfsDecadron Vincristine Delta-Cortef Vinorelbine Deltasone Vinorelbinetartrate Denileukin diftitox VLB DepoCyt VP-16 Dexamethasone VumonDexamethasone acetate Xeloda dexamethasone sodium Zanosar phosphateZevalin Dexasone Zinecard Dexrazoxane Zoladex DHAD Zoledronic acid DICZometa Diodex Gliadel wafer Docetaxel Glivec Doxil GM-CSF DoxorubicinGoserelin Doxorubicin liposomal granulocyte-colony stimulating factorDroxia Granulocyte macrophage DTIC colony stimulating factor DTIC-DomeHalotestin Duralone Herceptin Efudex Hexadrol Eligard Hexalen EllenceHexamethylmelamine Eloxatin HMM Elspar Hycamtin Emcyt Hydrea EpirubicinHydrocort Acetate Epoetin alfa Hydrocortisone Erbitux Hydrocortisonesodium phosphate Erwinia L-asparaginase Hydrocortisone sodium succinateEstramustine Hydrocortone phosphate Ethyol Hydroxyurea EtopophosIbritumomab Etoposide Ibritumomab Tiuxetan Etoposide phosphate IdamycinEulexin Idarubicin Evista Ifex Exemestane IFN-alpha Fareston IfosfamideFaslodex IL-2 Femara IL-11 Filgrastim Imatinib mesylate FloxuridineImidazole Carboxamide Fludara Interferon alfa Fludarabine InterferonAlfa-2b (PEG conjugate) Fluoroplex Interleukin-2 FluorouracilInterleukin-11 Fluorouracil (cream) Intron A (interferon alfa-2b)Fluoxymesterone Leucovorin Flutamide Leukeran Folinic Acid Leukine FUDRLeuprolide Fulvestrant Leurocristine G-CSF Leustatin Gefitinib LiposomalAra-C Gemcitabine Liquid Pred Gemtuzumab ozogamicin Lomustine GemzarL-PAM Gleevec L-Sarcolysin Lupron Meticorten Lupron Depot MitomycinMatulane Mitomycin-C Maxidex Mitoxantrone Mechlorethamine M-PrednisolMechlorethamine MTC Hydrochlorine MTX Medralone Mustargen Medrol MustineMegace Mutamycin Megestrol Myleran Megestrol Acetate Iressa MelphalanIrinotecan Mercaptopurine Isotretinoin Mesna Kidrolase Mesnex LanacortMethotrexate L-asparaginase Methotrexate Sodium LCR Methylprednisolone

TABLE 15 Examples of Cancer Types Acute Lymphoblastic Leukemia, AdultHairy Cell Leukemia Acute Lymphoblastic Leukemia, Head and Neck CancerChildhood Hepatocellular (Liver) Cancer, Adult (Primary) Acute MyeloidLeukemia, Adult Hepatocellular (Liver) Cancer, Childhood Acute MyeloidLeukemia, Childhood (Primary) Adrenocortical Carcinoma Hodgkin'sLymphoma, Adult Adrenocortical Carcinoma, Childhood Hodgkin's Lymphoma,Childhood AIDS-Related Cancers Hodgkin's Lymphoma During PregnancyAIDS-Related Lymphoma Hypopharyngeal Cancer Anal Cancer Hypothalamic andVisual Pathway Glioma, Astrocytoma, Childhood Cerebellar ChildhoodAstrocytoma, Childhood Cerebral Intraocular Melanoma Basal CellCarcinoma Islet Cell Carcinoma (Endocrine Pancreas) Bile Duct Cancer,Extrahepatic Kaposi's Sarcoma Bladder Cancer Kidney (Renal Cell) CancerBladder Cancer, Childhood Kidney Cancer, Childhood Bone Cancer,Osteosarcoma/Malignant Laryngeal Cancer Fibrous Histiocytoma LaryngealCancer, Childhood Brain Stem Glioma, Childhood Leukemia, AcuteLymphoblastic, Adult Brain Tumor, Adult Leukemia, Acute Lymphoblastic,Childhood Brain Tumor, Brain Stem Glioma, Leukemia, Acute Myeloid, AdultChildhood Leukemia, Acute Myeloid, Childhood Brain Tumor, CerebellarAstrocytoma, Leukemia, Chronic Lymphocytic Childhood Leukemia, ChronicMyelogenous Brain Tumor, Cerebral Leukemia, Hairy CellAstrocytoma/Malignant Glioma, Lip and Oral Cavity Cancer Childhood LiverCancer, Adult (Primary) Brain Tumor, Ependymoma Childhood Liver Cancer,Childhood (Primary) Brain Tumor, Medulloblastoma, Lung Cancer, Non-SmallCell Childhood Lung Cancer, Small Cell Brain Tumor, SupratentorialPrimitive Lymphoma, AIDS-Related Neuroectodermal Tumors, ChildhoodLymphoma, Burkitt's Brain Tumor, Visual Pathway and Lymphoma, CutaneousT-Cell, see Mycosis Hypothalamic Glioma, Childhood Fungoides and SézarySyndrome Brain Tumor, Childhood Lymphoma, Hodgkin's, Adult Breast CancerLymphoma, Hodgkin's, Childhood Breast Cancer, Childhood Lymphoma,Hodgkin's During Pregnancy Breast Cancer, Male Lymphoma, Non-Hodgkin's,Adult Bronchial Adenomas/Carcinoids, Lymphoma, Non-Hodgkin's, ChildhoodChildhood Lymphoma, Non-Hodgkin's During Pregnancy Burkitt's LymphomaLymphoma, Primary Central Nervous System Carcinoid Tumor, ChildhoodMacroglobulinemia, Waldenström's Carcinoid Tumor, GastrointestinalMalignant Fibrous Histiocytoma of Carcinoma of Unknown PrimaryBone/Osteosarcoma Central Nervous System Lymphoma, Medulloblastoma,Childhood Primary Melanoma Cerebellar Astrocytoma, Childhood Melanoma,Intraocular (Eye) Cerebral Astrocytoma/Malignant Glioma, Merkel CellCarcinoma Childhood Mesothelioma, Adult Malignant Cervical CancerMesothelioma, Childhood Childhood Cancers Metastatic Squamous NeckCancer with Occult Chronic Lymphocytic Leukemia Primary ChronicMyelogenous Leukemia Multiple Endocrine Neoplasia Syndrome, ChronicMyeloproliferative Disorders Childhood Colon Cancer MultipleMyeloma/Plasma Cell Neoplasm Colorectal Cancer, Childhood MycosisFungoides Cutaneous T-Cell Lymphoma, see Myelodysplastic SyndromesMycosis Fungoides and Sézary Myelodysplastic/Myeloproliferative DiseasesSyndrome Myelogenous Leukemia, Chronic Endometrial Cancer MyeloidLeukemia, Adult Acute Ependymoma, Childhood Myeloid Leukemia, ChildhoodAcute Esophageal Cancer Myeloma, Multiple Esophageal Cancer, ChildhoodMyeloproliferative Disorders, Chronic Ewing's Family of Tumors NasalCavity and Paranasal Sinus Cancer Extracranial Germ Cell Tumor,Nasopharyngeal Cancer Childhood Nasopharyngeal Cancer, ChildhoodExtragonadal Germ Cell Tumor Neuroblastoma Extrahepatic Bile Duct CancerNon-Hodgkin's Lymphoma, Adult Eye Cancer, Intraocular MelanomaNon-Hodgkin's Lymphoma, Childhood Eye Cancer, RetinoblastomaNon-Hodgkin's Lymphoma During Pregnancy Gallbladder Cancer Non-SmallCell Lung Cancer Gastric (Stomach) Cancer Oral Cancer, Childhood Gastric(Stomach) Cancer, Childhood Oral Cavity Cancer, Lip and GastrointestinalCarcinoid Tumor Oropharyngeal Cancer Germ Cell Tumor, Extracranial,Osteosarcoma/Malignant Fibrous Histiocytoma Childhood of Bone Germ CellTumor, Extragonadal Ovarian Cancer, Childhood Germ Cell Tumor, OvarianOvarian Epithelial Cancer Gestational Trophoblastic Tumor Ovarian GermCell Tumor Glioma, Adult Ovarian Low Malignant Potential Tumor Glioma,Childhood Brain Stem Pancreatic Cancer Glioma, Childhood CerebralPancreatic Cancer, Childhood Astrocytoma Pancreatic Cancer, Islet CellGlioma, Childhood Visual Pathway and Paranasal Sinus and Nasal CavityCancer Hypothalamic Parathyroid Cancer Skin Cancer (Melanoma) PenileCancer Skin Carcinoma, Merkel Cell Pheochromocytoma Small Cell LungCancer Pineoblastoma and Supratentorial Primitive Small Intestine CancerNeuroectodermal Tumors, Childhood Soft Tissue Sarcoma, Adult PituitaryTumor Soft Tissue Sarcoma, Childhood Plasma Cell Neoplasm/MultipleMyeloma Squamous Cell Carcinoma, see Skin Pleuropulmonary BlastomaCancer (non-Melanoma) Pregnancy and Breast Cancer Squamous Neck Cancerwith Occult Pregnancy and Hodgkin's Lymphoma Primary, MetastaticPregnancy and Non-Hodgkin's Lymphoma Stomach (Gastric) Cancer PrimaryCentral Nervous System Lymphoma Stomach (Gastric) Cancer, ChildhoodProstate Cancer Supratentorial Primitive Rectal Cancer NeuroectodermalTumors, Childhood Renal Cell (Kidney) Cancer T-Cell Lymphoma, Cutaneous,see Renal Cell (Kidney) Cancer, Childhood Mycosis Fungoides and SézaryRenal Pelvis and Ureter, Transitional Cell Syndrome Cancer TesticularCancer Retinoblastoma Thymoma, Childhood Rhabdomyosarcoma, ChildhoodThymoma and Thymic Carcinoma Salivary Gland Cancer Thyroid CancerSalivary Gland Cancer, Childhood Thyroid Cancer, Childhood Sarcoma,Ewing's Family of Tumors Transitional Cell Cancer of the Renal Sarcoma,Kaposi's Pelvis and Ureter Sarcoma, Soft Tissue, Adult TrophoblasticTumor, Gestational Sarcoma, Soft Tissue, Childhood Unknown Primary Site,Carcinoma of, Sarcoma, Uterine Adult Sezary Syndrome Unknown PrimarySite, Cancer of, Skin Cancer (non-Melanoma) Childhood Skin Cancer,Childhood Unusual Cancers of Childhood Ureter and Renal Pelvis,Transitional Cell Cancer Urethral Cancer Uterine Cancer, EndometrialUterine Sarcoma Vaginal Cancer Visual Pathway and Hypothalamic Glioma,Childhood Vulvar Cancer Waldenström's Macroglobulinemia Wilms' Tumor

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues. For example, a particular cancer may becharacterized by a solid tumor mass or a non-solid tumor. A primarytumor mass refers to a growth of cancer cells in a tissue resulting fromthe transformation of a normal cell of that tissue. In most cases, theprimary tumor mass is identified by the presence of a cyst, which can befound through visual or palpation methods, or by irregularity in shape,texture, or weight of the tissue. However, some primary tumors are notpalpable and can be detected only through medical imaging techniquessuch as X-rays (e.g., mammography), or by needle aspirations. The use ofthese latter techniques is more common in early detection. Molecular andphenotypic analysis of cancer cells within a tissue will usually confirmif the cancer is endogenous to the tissue or if the lesion is due tometastasis from another site. Depending upon the type of agent (payload)utilized, the compositions of the invention may be capable of inducingapoptosis in tumor cells and reducing tumor cell growth. Thecompositions of the invention can be administered locally at the site ofa tumor (e.g., by direct injection) or remotely. Depending upon thepayload, the compositions of the invention can induce cell death incirculating tumor cells (CTC) in a subject, e.g., by administering thecompositions intravenously. Furthermore, depending upon payload, thecompositions of the invention can prevent or reduce onset of metastasisto other tissues. Furthermore, in cases in which the payload is adetectable moiety, such as a contrast agent, the compositions of theinvention can be used to detect metastasis to other tissues andpotentially avoid the need for nodal biopsy.

As used herein, the term “payload” refers to agents and moieties linkedto the MC1R peptide ligand or residing within the inner core of the MC1Rpeptide ligand-micelle complex. The payload may be any desired agent ormoiety that is capable of being directly or indirectly linked to theMC1R peptide ligand or incorporated within the micelle. Examples ofpayloads include, but are not limited to, molecules such as contrastagents (e.g., detectable substances such as dyes), biologically activeagents, such as biologics, anti-cancer agents such as chemotherapeuticagents (see, for example, Table 14), or other drugs. The terms“payload”, “agent”, and “moiety” are used interchangeably herein.

As used in this specification, the singular forms “a”, “an”, and “the”include plural reference unless the context clearly dictates otherwise.Thus, for example, a reference to “a cell” includes one or more cells. Areference to “a peptide” includes one or more such peptide, and soforth.

The practice of the present invention can employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology, electrophysiology, and pharmacology that arewithin the skill of the art. Such techniques are explained fully in theliterature (see, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning:A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II(D. N. Glover Ed. 1985); Perbal, B., A Practical Guide to MolecularCloning (1984); the series, Methods In Enzymology (S. Colowick and N.Kaplan Eds., Academic Press, Inc.); Transcription and Translation (Hameset al. Eds. 1984); Gene Transfer Vectors For Mammalian Cells (J. H.Miller et al. Eds. (1987) Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.); Scopes, Protein Purification: Principles and Practice(2nd ed., Springer-Verlag); and PCR: A Practical Approach (McPherson etal. Eds. (1991) IRL Press)), each of which are incorporated herein byreference in their entirety.

EXEMPLIFIED EMBODIMENTS Embodiment 1

A modified melanocortin 1 receptor (MC1R) peptide ligand, comprising anMC1R peptide ligand coupled to a functionality.

Embodiment 2

The modified MC1R peptide ligand of embodiment 1, wherein thefunctionality is an alkyne, azide, amine, aldehyde, thiol, alkene,ester, or maleimide.

Embodiment 3

The modified MC1R peptide ligand of embodiment 1, wherein thefunctionality is coupled to the C-terminus of said MC1R ligand.

Embodiment 4

The modified MC1R peptide ligand of embodiment 1, wherein thefunctionality is coupled to the N-terminus of said MC1R ligand.

Embodiment 5

The modified MC1R peptide ligand of embodiment 1, wherein the MC1Rpeptide ligand is selected from:

(SEQ ID NO: 3) 4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂; (SEQ ID NO: 4)H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂;  or  (SEQ ID NO: 5)H-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂. 

Embodiment 6

The modified MC1R peptide ligand of embodiment 1, wherein the MC1Rpeptide ligand comprises the amino acid motif His-Phe-Arg-Trp (HFRW)(SEQ ID NO:1).

Embodiment 7

The modified MC1R peptide ligand of embodiment 1, wherein the MC1Rpeptide ligand comprises the amino acid motif His-DPhe-Arg-Trp (HfRW)(SEQ ID NO:2).

Embodiment 8

A method of preparing a MC1R peptide ligand according to embodiment 1,comprising:

-   -   providing an MC1R peptide ligand; and    -   covalently bonding a moiety comprising a functionality to the        C-terminus or N-terminus of the MC1R peptide ligand, wherein a        MC1R peptide ligand comprising a functionality is formed.

Embodiment 9

The method of embodiment 8, wherein the MC1R peptide ligand comprises aLys residue or other nitrogen-bearing, thiol-bearing, or —OH bearingresidue at the C-terminus or N-terminus, and wherein the moietycomprising the functionality is covalently bound to the residue.

Embodiment 10

The method of embodiment 9, wherein the residue at the C-terminus orN-terminus comprises the Lys residue.

Embodiment 11

The method of embodiment 8, wherein the moiety comprises a terminalalkynyl acid of 5 to 12 carbons.

Embodiment 12

A melanocortin 1 receptor (MC1R)-targeted agent comprising:

-   -   a MC1R peptide ligand; and    -   a moiety, wherein the MC1R peptide ligand and the moiety are        covalently linked by a reaction product of a first functionality        coupled to the peptide ligand and a complementary second        functionality coupled to the moiety.

Embodiment 13

The melanocortin 1-targeted agent of embodiment 12, wherein the moietycomprises an anti-cancer agent, drug (e.g., chemotherapeutic agent,immunotherapeutic agent, etc.), contrast agent, polymer, gel, particle,surface, or any combination of one or more of the foregoing.

Embodiment 14

The melanocortin 1-targeted agent of embodiment 13, wherein the moietycomprises a contrast agent comprising a fluorescent dye.

Embodiment 15

The melanocortin 1-targeted agent of embodiment 14, wherein thefluorescent dye comprises RDye800CW.

Embodiment 16

The melanocortin 1-targeted agent of any one of embodiment 13-15,wherein the contrast agent is covalently linked to the N terminus of theMC1R peptide ligand.

Embodiment 17

The melanocortin 1-targeted agent of embodiment 13 or 14, wherein thecontrast agent is covalently linked to the C terminus of the MC1Rpeptide ligand.

Embodiment 18

The melanocortin 1-targeted agent of embodiment 12, wherein the firstfunctionality comprises an alkyne, azide, amine, aldehyde, thiol,alkene, ester, or maleimide and the reaction product is a1,2,3-triazole, imine, disulfide, thioether, primary amide, or secondaryamide.

Embodiment 19

A method of delivering a moiety to cells expressing the melanocortin 1receptor (MC1R), comprising administering an MC1R-targeted agent ofembodiment 12 or 13 to the cells in vitro or in vivo.

Embodiment 20

The method of embodiment 19, wherein the MC1R-targeted agent isadministered to the cells in vivo.

Embodiment 21

The method of embodiment 20, wherein the MC1R-targeted agent isadministered systemically to a human or non-human animal subject.

Embodiment 22

The method of embodiment 21, wherein the MC1R-targeted agent isadministered to the subject intravascularly (e.g., intravenously).

Embodiment 23

The method of embodiment 20, wherein the MC1R-targeted agent isadministered locally to the cells.

Embodiment 24

The method of embodiment 19, wherein the MC1R-targeted agent isadministered to a human or non-human animal subject, and wherein themoiety comprises a contrast agent.

Embodiment 25

The method of embodiment 20, further comprising imaging the subjectusing an imaging modality.

Embodiment 26

The method of embodiment 19, wherein the MC1R-targeted agent isadministered locally or systemically to a human or non-human animalsubject having melanoma, and wherein the moiety comprises an anti-canceragent.

Embodiment 27

The method of embodiment 26, wherein the anti-cancer agent kills orinhibits the growth of melanoma cells.

Embodiment 28

A pharmaceutical composition comprising the MC1-R targeted agent ofembodiment 12 or 13, and a pharmaceutically acceptable carrier.

Embodiment 29

A method of treating melanoma in a human or non-human animal subject,comprising administering an MC1R-targeted agent of embodiment 12 or 13to the subject, wherein the moiety comprises an anti-cancer agent.

Embodiment 30

The method of embodiment 29, wherein the anti-cancer agent kills orinhibits the growth of melanoma cells.

Embodiment 31

A modified melanocortin 1 receptor (MC1R) peptide ligand-micellecomplex, comprising:

-   -   an MC1R peptide ligand; and    -   a micelle comprising an inner core, outer core and hydrophilic        shell, wherein the MC1R peptide ligand is linked to the shell of        the micelle by a linker.

Embodiment 32

The MC1R peptide ligand-micelle complex of embodiment 31, furthercomprising an agent residing in the inner core of the micelle.

Embodiment 33

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe linker comprises a 1,2,3-triazole, imine, disulfide, thioether,primary amide, or secondary amide.

Embodiment 34

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe inner core of the micelle comprises a hydrophobic polypeptide, theouter core comprises a crosslinked peptide comprising a multiplicity ofcrosslinked amino acid residues and the hydrophilic shell comprises awater soluble polymer, and wherein the inner core is covalently attachedto the outer core and the outer core is covalently attached to thehydrophilic shell.

Embodiment 35

The MC1R peptide ligand-micelle complex of embodiment 34, wherein thewater soluble polymer comprises polyethylene glycol.

Embodiment 36

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe linker is a 1,2,3-triazole from the addition of an azide and analkyne.

Embodiment 37

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe linker is coupled to the C-terminus of said MC1R ligand.

Embodiment 38

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe linker is coupled to the N-terminus of said MC1R ligand.

Embodiment 39

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe linker comprises a reaction product, and wherein the MC1R peptideligand is selected from:

(SEQ ID NO: 3) 4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂;  (SEQ ID NO: 4)H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂; or  (SEQ ID NO: 5)H-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)- Arg-DPhe-Asp-Arg-Phe-Gly-NH₂.

Embodiment 40

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe MC1R peptide ligand comprises the amino acid motif His-Phe-Arg-Trp(HFRW) (SEQ ID NO:1).

Embodiment 41

The MC1R peptide ligand-micelle complex of embodiment 31 or 32, whereinthe MC1R peptide ligand comprises the amino acid motif His-DPhe-Arg-Trp(HfRW) (SEQ ID NO:2).

Embodiment 42

The MC1R peptide ligand-micelle complex of embodiment 32, wherein theagent comprises an anti-cancer drug.

Embodiment 43

The MC1R peptide ligand-micelle complex of embodiment 32, wherein theagent comprises a contrast agent.

Embodiment 44

A method of imaging a melanoma tumor of a subject, comprising:

-   -   administering the MC1R peptide ligand-micelle complex of        embodiment 31 or 32 to the subject, wherein a contrast agent is        present within the inner core of the micelle, and wherein the        MC1R peptide ligand-micelle complex concentrates in the tumor;        and    -   observing a signal from the contrast agent, e.g., using an        imaging device also referred to herein as an imaging modality).

Embodiment 45

A method of imaging a melanoma tumor, comprising:

-   -   providing a MC1R peptide ligand-micelle complex of embodiment 31        or 32;    -   incorporating a contrast agent into the inner core of said MC1R        peptide ligand micelle complex;    -   administering the MC1R peptide ligand-micelle complex with the        contrast agent to a human or non-human animal subject, wherein        the MC1R peptide ligand-micelle complex concentrates in the        tumor; and    -   observing a signal from the contrast agent, e.g., using an        imaging device (also referred to herein as an imaging modality).

Embodiment 46

The method of embodiment 45, wherein the contrast agent is a nearinfrared (NIR) fluorescent dye.

Embodiment 47

The method of embodiment 46, wherein the NIR fluorescent dye comprises aICG derivative.

Embodiment 48

The method of embodiment 45, wherein the contrast agent is a CT contrastagent.

Embodiment 49

The method of embodiment 48, wherein the CT contrast agent comprisesgold.

Embodiment 50

The method of embodiment 45, wherein the contrast agent is a MRI orSPECT contrast agent.

Embodiment 51

The method of embodiment 50, wherein the MRI or SPECT contrast agentcomprises Gd, Tc99m, or an ¹¹¹In chelate.

Embodiment 52

The method of embodiment 45, wherein the contrast agent is a PET imagingagent.

Embodiment 53

The method of embodiment 52, wherein the PET imaging agent comprises18-F, 11-C, 18-O, or Gallium 64.

Embodiment 54

A method of treating melanoma tumor cells in a subject, comprising:

-   -   administering the MC1R peptide ligand-micelle complex of        embodiment 31 or 32, to the subject, wherein an anti-cancer        agent is present within the inner core of the micelle, and        wherein the MC1R peptide ligand-micelle complex releases the        anti-cancer agent at the site of the tumor.

Embodiment 55

A method of treating melanoma tumor cells in a subject, comprising:

-   -   providing a MC1R peptide ligand-micelle complex of embodiment 31        or 32;    -   incorporating an anti-cancer agent into the inner core of said        MC1R peptide ligand-micelle complex;    -   administering the MC1R peptide ligand-micelle complex with the        anti-cancer agent to the subject, wherein the MC1R peptide        ligand-micelle complex releases the anti-cancer agent at the        site of the tumor.

Embodiment 56

The method of embodiment 55, wherein the anti-cancer agent is aradiotherapy agent.

Embodiment 57

The method of embodiment 56, wherein the radiotherapy agent comprisesYttrium.

Embodiment 58

The method of embodiment 55, wherein the anti-cancer agent comprises analkylating chemotherapy agent.

Embodiment 59

The method of embodiment 58, wherein the alkylating chemotherapy agentcomprises melphalan or ifosfamide.

Embodiment 60

The method of embodiment 55, wherein the anti-cancer agent is a systemicmelanoma chemotherapy agent.

Embodiment 61

The method of embodiment 60, wherein the systemic melanoma chemotherapyagent comprises dacarbazine, paclitaxel, and/or vincristine.

Embodiment 62

A method of preparing a MC1R peptide ligand-micelle complex, comprising:

-   -   providing a MC1R peptide ligand comprising a first functionality        covalently attached to a MC1R targeting peptide;    -   providing a multiplicity of triblock polymer chains comprising a        hydrophobic polypeptide block attached to a central        crosslinkable peptide block comprising a multiplicity of        crosslinkable amino acid residues attached to a water soluble        polymer block, wherein a portion of the triblock polymer chains        further comprise a second functionality covalently attached to        the water soluble polymer block distal to the central        crosslinkable peptide block, wherein the second functionality is        complementary to the first functionality and wherein the        triblock polymer chains self-assemble into a micelle; and    -   combining the triblock polymer chains with the MC1R peptide        ligand, wherein the first functionality and the complementary        second click functionality react to form a reaction product that        covalently joins the triblock polymer to the MC1R peptide ligand        to form a MC1R peptide ligand-micelle complex.

Embodiment 63

The method of embodiment 62, wherein the MC1R peptide ligand is4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂ (SEQ ID NO:3);H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂(SEQ ID NO:4); orH-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂(SEQ ID NO:5).

Embodiment 64

The method of embodiment 62 or 63, further comprising crosslinking themultiplicity of crosslinkable amino acid residues.

Embodiment 65

The method of embodiment 62 or 63, further comprising the incorporationof an agent.

Embodiment 66

The method of embodiment 65, wherein the agent comprises a contrastagent, anti-cancer agent, or both.

Embodiment 67

A pharmaceutical composition comprising the MC1R peptide ligand-micellecomplex of embodiment 31 or 32; and a pharmaceutically acceptablecarrier.

Embodiment 68

The MC1R peptide ligand, method, composition, or MC1R peptideligand-micelle complex of any one of embodiments 1-67, wherein the agentis a biologically active agent or contrast agent.

Embodiment 69

The MC1R peptide ligand, method, composition, or MC1R peptideligand-micelle complex of embodiment 68, wherein the biologically activeagent is a drug or biologic.

Embodiment 70

The MC1R peptide ligand, method, composition, or MC1R peptideligand-micelle complex of embodiment 68, wherein the biologically activeagent is an anti-cancer agent.

Embodiment 71

The MC1R peptide ligand, method, composition, or MC1R peptideligand-micelle complex of embodiment 70, wherein the anti-cancer agentis an immunotherapeutic agent, a chemotherapeutic agent listed in Table14, or another chemotherapeutic agent.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims. In addition, anyelements or limitations of any invention or embodiment thereof disclosedherein can be combined with any and/or all other elements or limitations(individually or in any combination) or any other invention orembodiment thereof disclosed herein, and all such combinations arecontemplated with the scope of the invention without limitation thereto.

All patents, patent applications, provisional applications, andpublications referred to or cited herein, supra or infra, areincorporated by reference in their entirety, including all figures andtables, to the extent they are not inconsistent with the explicitteachings of this specification.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Materials and Methods for Example 1

Abbreviations used for amino acids and designation of peptides followthe rules of the IUPAC-IUB Commission of Biochemical Nomenclature in J.Biol. Chem. 1972, 247:977-983. The following additional abbreviationsare used: Aloc, allyloxycarbonyl; Boc, tert-butyloxycarbonyl; ^(t)Bu,tert-butyl; DMSO, dimethylsulfoxide; DVB, divinylbenzene; Fmoc,(9H-fluoren-9-ylmethoxy)carbonyl; HBTU,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluoro-phosphate; HOBt, N-hydroxybenzotriazole; HOCt,6-chloro-1H-hydroxybenzotriazole; NMI, N-methylimidazole; Pbf,2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl; PS, polystyrene;RP-HPLC, reverse-phase high performance liquid chromatography; TFA,trifluoroacetic acid; and Trt, triphenylmethyl (trityl).

Materials.

N-α-Fmoc-protected amino acids, HBTU, HCTU, HOCt and HOBt were purchasedfrom Anaspec (San Jose, Calif.) or from Novabiochem (San Diego, Calif.).Rink amide Tentagel S and R resins were acquired from Rapp Polymere(Tubingen, Germany). Rink amide 1%-DVB PS resin was acquired fromNovabiochem (San Diego, Calif.). For the N-α-Fmoc-protected amino acids,the following side chain protecting groups were used: Arg(N^(g)-Pbf);Asp(O-tBu); His(N^(im)-Trt); Trp(N^(i)-Boc); Tyr(^(t)Bu), andLys(N^(ε)-Aloc). IRDye 800CW maleimide was acquired from Li-Cor(Lincoln, Nebr.). Reagent grade solvents, reagents, and acetonitrile(ACN) for HPLC were acquired from VWR (West Chester, Pa.) orAldrich-Sigma (Milwaukee, Wis.), and were used without furtherpurification unless otherwise noted. N-terminal heterocyclic acids, NMI,and scavengers were obtained from Sigma-Aldrich or TCI. The solid-phasesynthesis was performed in fitted syringes using a Domino manualsynthesizer obtained from Torviq (Niles, Mich.). The C-18 Sep-Pak™ VacRC cartridges for solid phase extraction were purchased from Waters(Milford, Mass.).

Ligand Synthesis.

Ligands 1-8 were prepared as previously published by solid-phasesynthesis as summarized in Scheme 1 on Rink Amide Tentagel resin (0.23mmol/g) using a Fmoc/^(t)Bu synthetic strategy and standardactivations.[30, 31] After final deprotection of the Fmoc group, theresin was coupled with HOBt ester of 4-phenylbutyric acid (compounds 1,4), acetylated with acetic anhydride/pyridine (compound 2) or leftunreacted as a free amino group (compounds 5-8). The4-hydroxycinnamoyl-His-DPhe-Arg-Trp-NH (SEQ ID NO:8) NH-resin wastreated with 50% piperidine in DMF to remove 4-hydroxycinnamoyloligomers. The ligands were cleaved off the resins with TFA-scavengercocktail (91% TFA, 3% water, 3% thioanisole, 3% ethanedithiol),extracted with cold diethylether, then dissolved in 1.0 m aqueous aceticacid. The crude ligands were purified by SEC and HPLC. The purecompounds were dissolved in DI water or DMSO at approximately 1.0 mMconcentrations and concentration was determined by Trp-HPLCmeasurement[32, 33].

Ligand Purification and Characterization.

Peptides were purified using solid-phase extraction. Briefly, C-18Sep-Pak™ cartridges (100 mg or 500 mg) were used and pre-conditionedinitially with acetonitrile, methanol, and water. After loading thecompound, the column was washed with DI water, and then gradually with5, 10, 20, 30, 50 and 70% of aqueous ACN. Fractions containing productwere collected, concentrated to remove organic solvent and lyophilized.Product purity was verified by analytical RP-HPLC using a WatersAlliance 2695 Separation Model with a Waters 2487 dual wavelengthdetector (220 and 280 nm) on a reverse phase column (Waters XBridge C18,3.0×75 mm, 3.5 mm). Peptides were eluted with a linear gradient ofaqueous ACN/0.1% TFA at a flow rate of 0.3 mL/min. Purification ofligands was achieved on a Waters 600 HPLC using a reverse phase column(Waters)(Bridge C18, 19.0×250 mm, 10 mm). Peptides were eluted with alinear gradient of ACN/0.1% TFA at a flow rate of 5.0-10.0 mL/min.Separation was monitored at 230 and 280 nm. Size exclusionchromatography was performed on a borosilicate glass column (2.6×250 mm,Sigma, St. Louis, Mo.) filled with medium sized Sephadex G-25 or G-10.The compounds were eluted with an isocratic flow of 1.0 M aqueous aceticacid. Mass spectra and HPLC characterization is given in Table 2, below.

QC and Purification:

The purity of products was checked by analytical PR-HPLC using a WatersAlliance 2695 Separation Model with a Waters 2487 dual wavelengthdetector (220 and 280 nm) on a reverse phase column (Waters)(Bridge C18,3.0×75 mm, 3.5 μm). Peptides were eluted with a linear gradient ofaqueous ACN/0.1% TFA at a flow rate of 0.3 mL/min. Purification ofligands was achieved on a Waters 600 HPLC using a reverse phase column(Waters)(Bridge C18, 19.0×250 mm, 10 μm). Peptides were eluted with alinear gradient of ACN/0.1% TFA at a flow rate of 5.0-10.0 mL/min.Separation was monitored at 230 and 280 nm. Size exclusionchromatography was performed on a borosilicate glass column (2.6×250 mm,Sigma, St. Louis, Mo.) filled with medium sized Sephadex G-25 or G-10.The compounds were eluted with an isocratic flow of 1.0 M aqueous aceticacid.

Solid-Phase Extraction (SPE): C-18 Sep-Pak™ cartridges (100 mg or 500mg) were used and pre-conditioned initially with 5 column volumes (5times the volume of packed column bed) each of acetonitrile, methanol,and water. After loading the compound, the column was washed with DIwater, and then gradually with 5, 10, 20, 30, 50, and 70% of aqueousACN. Fraction containing product were collected, concentrated to removeorganic solvent, and lyophilized.

Quantitative HPLC: The peptide concentrations were determined bymonitoring absorbance of peptides against 0.5 mM solution of Tryptophanin DMSO at 280 nm. The peptides were initially dissolved in DMSO atapproximately 1-5 mM concentration. Co-injections of peptide and Trpwere made on analytical HPLC with a number of different volumes andpeptide concentration calculated from area under the peaks using theformula given here:

${{Peptide}\mspace{14mu}{{Conc}.}} = {\frac{\left\lbrack {{{Abs}.\mspace{14mu}{of}}\mspace{14mu}{{Comp}.}} \right\rbrack}{\left\lbrack {{{Abs}.\mspace{14mu}{of}}\mspace{14mu}{Trp}} \right\rbrack} \times \frac{0.5}{\frac{\sum{ɛ_{280}\left( {{Trp} + {Tyr} + {Cys} + {{Cy}\; 5} + \ldots} \right)}}{ɛ_{280}}} \times \frac{{{Vol}.\mspace{14mu}{of}}\mspace{14mu}{Trp}}{{{Vol}.\mspace{14mu}{of}}\mspace{14mu}{{Comp}.}}}$ε₂₈₀ of compounds were determined by summation of tryptophans(ε₂₈₀=5500), tyrosine (ε₂₈₀=1490), thiol (ε₂₈₀=63), and Cy5 dye(ε₂₈₀=5800), and normalized to extinction coefficient of one tryptophan.Other amino acids in these peptides do not absorb significantly at thiswavelength. For Cy5 dye, ε₂₈₀ was determined in a similar manner bycomparing the absorbances of 0.5 mM of both Trp and Cy5-malenimide esterin DMSO at 280 nm wavelength. Mass spectra and HPLC characterizationdata are provided in Table 2, below.

Mass Spectrometry:

Mass spectra of positive ions were recorded either with a single stagereflectron MALDI-TOF mass spectrometer (Bruker Rexlex III, BrukerDaltonics, Billerica, Mass.; α-cyanocinnamic acid as a matrix) inreflectron mode or with a low resolution ESI mass spectrometer(Finnigan, Thermoquest LCQ ion trap instrument, Lake Forrest, Calif.)and/or using high resolution Fourier transform mass spectrometer (FT-ICRMS, Bruker Apex Qh, Bremen, Germany) equipped with an ESI source. Forinternal calibration, an appropriate mixture of standard peptides wasused with an average resolution of ca. 10,000 on the Reflex III and60,000 on the FT-ICR instrument.

TABLE 2 High resolution mass spectral data and HPLC^(a) Compound R_(t)(k′) Calc. [MH⁺] Exp. [MH⁺] 1 4.14 790.4147 790.5 2 3.89 746.4362 746.53 2.54 790.3783 790.4 4 4.32 1069.5730 1069.5726 5 3.61 1601.8124 1601.86 3.90 1823.9493 1824.0 7 3.41 1562.8015 1562.5 8 3.79 1784.9384 1784.3^(a)Peptide was eluted with a linear ACN/0.1% CF₃CO₂H aqueous gradient(10% to 90% in 30 min) at a flow rate of 0.3 mL/min); Waters XBridgeC-18 column (3.0 × 150 mm, 3.5 μm); HPLC k′ = (peptide retentiontime-solvent retention time)/solvent retention time. All the obtainedpurified peptides showed >95% purity. [b] The major molecular peakcorresponds to [M + 3Na]⁺, formula C₁₀₅H₁₂₇O₂₄S₅N₁₈Na₃.

Cell Culture.

HCT116 cells overexpressing hMC1R and HEK293 cells overexpressinghMC4R[23] or hMC5R were used in all studies. The parental humancolorectal carcinoma cell line, HCT116 (American Type CultureCollection, CCL 247) was also used. Cells were maintained under standardconditions (37° C. and 5% CO₂) and were grown in Dulbecco's modifiedEagle medium (DMEM) supplemented with 10% FBS and 5%penicillin/streptomycin. For HCT116/hMC1R cells, geneticin (G418S, 0.4mg/mL) was added to the media to ensure proper selection.

Europium Binding Assays.

Competitive binding assays were performed using HCT116/hMC1R cells andHEK293/hMC4R or hMC5R as previously described, with slightmodifications[23]. HCT116/hMC1R cells were plated in black PerkinElmer96-well plates and HEK293/hMC4R and HEK293/hMC5R cells were plated onSigmaScreen Poly-D-Lysine Coated Plates (SigmaAldrich), all at a densityof 10,000-30,000 cells/well. Poly-D-Lysine Coated Plates contain a PDLpolymer coating that creates a uniform positive charge at the surface ofthe plastic, thereby facilitating cell attachment, growth anddifferentiation. Cells were grown in the 96-well plates for 2-3 days. Onthe day of the experiment, the media was aspirated and 50 μL ofnon-labeled competing ligand was added to each well in a series ofdecreasing concentrations (ranging from ˜1 μM to 0.1 nM), followed by 50μL of Eu-NDP-α-MSH at 10 nM. Both labeled and non-labeled ligands werediluted in binding media (DMEM, 1 mM 1,10-phenanthroline, 200 mg/Lbacitracin, 0.5 mg/L leupeptin, 0.3% BSA). In the case of the triblockpolymer micelle solutions, micelles were allowed to equilibrate insolution for a period of 30 min prior to cell addition. Cells wereincubated with labeled and non-labeled ligands for 1 hour at 37° C.Following incubation, cells were washed three times with wash buffer (50mM Tris-HCl, 0.2% BSA, 30 mM NaCl) and 50 μL of enhancement solution(PerkinElmer) was added to each well. Cells were incubated for anadditional 30 min at 37° C. prior to reading. The plates were read on aPerkinElmer VICTORx4 2030 multilabel reader using the standard Eu TRFmeasurement (340 nm excitation, 400 s delay, and emission collection for400 s at 615 nm). Competition curves were analyzed with GraphPad Prismsoftware using the sigmoidal dose-response (variable slope) classicalequation for nonlinear regression analysis.

Synthesis of Targeted Triblock Polymers.

Triblock polymer with a terminal azide incorporated into thepolyethylene glycol (PEG) block were obtained from IntezyneTechnologies, LLC (Tampa, Fla.). The triblock polymer comprised a shellhydrophilic block of PEG, a outer core block of comprising Asp, and acore block comprising a hydrophobic block of Leu and Tyr. To a solutionof 1:1 DMSO:H2O (10 mL) was added 4 (16 mmol, 1.2 equiv), triblockpolymer (13.3 mmol, 1 equiv), sodium ascorbate (334.15 mmol, 25 equiv),(BimC₄A)₃ catalyst[34] (13.42 mmol, 1 equiv). The solution was heated to50° C. and stirred for 2 days. The mixture was then cooled and placed ina 3500 MW dialysis bag and dialyzed against EDTA/H2O (×3) and H2O (×3).Following purification by dialysis, the solution was lyophilized.Successful click coupling was verified through visualization of thetriazole-H in 1H NMR (8.02 ppm).

Micelle Formulation.

Triblock polymers were dissolved at 20 mg/mL in 30% tert-butanol/waterat room temperature, stirred for 4 hours and then lyophilized. For thetargeted micelle system, 10% targeted polymer and 90% untargeted polymerwere used in the formulation mixture. Micelle size was determined bydynamic light scattering (DLS) and surface charge was determined by zetameasurement.

Competitive Binding Assays.

Competitive binding assays were performed using HCT116/hMC1R,HEK293/hMC4R and HEK293/hMC5R cells with ligands 1-8. 1, 2 and 4 werefound to both strongly and selectively bind to the hMC1R receptor (FIG.3, Table 2), with 1 and 4 showing slightly higher affinities for hMC1Ras compared to 2 (K_(i) is 0.17 nM, 0.24 nM and 1.77 nM for 1, 4 and 2,respectively, against hMC1R), and the selectivity of 1 is slightlyhigher than that of 2. Ligands 5-8 demonstrated a high affinity forhMC1R as well; however, they were also shown to have an even strongeraffinity for hMC4R and hMC5R. Ligand 3 demonstrated no affinity tohMC1R.

TABLE 3 Affinity and selectivity of ligands assayed in this publication.Ki (nM)⁺ Values determined Values derived from literature Ligand MC1RMC4R MC5R 1R/4R 1R/5R MC1R MC4R MC5R 1R/4R 1R/5R 1  0.17 160.00 27.00946.75 159.76 0.01  7.30 NA 1216.67 NA 2  1.77 988.20 58.19 558.31 32.88 NA NA NA NA NA 3 NB^(┤) NB NB NA NA NA NA NA NA NA 4  0.24 NA^(∥)NA NA NA NA NA NA NA NA 5  1.98  0.75  0.76  0.38  0.38 0.30^(⊥) 6.00^(⊥) 3.50^(⊥)  20.00 11.67 6  2.59  1.74  1.48  0.67  0.57 NA NA NANA NA 7  5.64  0.77  0.71  0.13  0.13 1.60^(⊥) 20.00^(⊥) 3.30^(⊥)  12.50 2.06 8  4.19  4.40  3.90  1.05  0.93 NA NA NA NA NA 4-targeted 25.65 NANA NA NA NA NA NA NA NA polymer 4-targeted 11.47 NA NA NA NA NA NA NA NANA micelles NDP-a-MSH  1.80  18.80  9.90  10.44  5.50 0.40  3.50 NA  8.75 NA ⁺All values represent Ki values except where denoted. ^(┤)NB =Non-binding; ^(∥)NA = Not available; ^(⊥)Value denotes an EC50 (nM)value.

Competitive binding assays were also performed using 4-targeted triblockpolymers, triblock polymer micelles, as well as untargeted micelles as acontrol (FIG. 4A-C). The 4-targeted micelle exhibits an increasedbinding avidity to the hMC1R receptor compared to the free polymer(K_(i) is 11.47 nM versus 25.65 nM for the micelle and polymerrespectively), and a slightly weaker avidity than the native ligand(K_(i) is 11.47 nM versus 1.17 nM for the micelle and ligandrespectively). Not surprisingly, no binding is observed with theuntargeted micelles, indicating that the triblock polymer does notinteract non-specifically with the cell surface. DLS and zeta potentialmeasurements showed the size and surface charge of the 4-targetedmicelles to be 91 nm and 0.93 mV, respectively.

Example 1—Preparation of MC1R-Targeted Tri-Block Polymer Micelle

Historically, ligands which are known to interact with the hMC1Rreceptor also demonstrate cross-reactivity with other melanocortinreceptors, namely hMC4R and hMC5R. While MC1R is known to be expressedalmost exclusively in melanoma cells and melanocytes, hMC4R and hMC5Rhave high expression levels in normal tissues including kidney andbrain, thus non-specific ligand binding is not ideal. To combat thisproblem and minimize off-target effects, several ligands from theliterature were examined that have been reported to possess nanomolarbinding affinities for hMC1R (Table 3). Ligand 1 was reported to have ahigh affinity and selectivity for MC1R (1R/4R selectivity ratio of1200)[20, 28] and was consequently chosen as a template for the designof the novel ligands 2, 3 and 4. Additionally, ligands 5 and 7 werereported to have moderate hMC1R selectivities over hMC4R and hMC5R[22];thus, each was functionalized with a terminal alkyne for attachment to ananoparticle scaffold.

Literature reported dissociation constants for all ligands tested arelisted in Table 3 for comparison. The most specific of these ligands, 1,was determined to have an hMC1R/hMC4R selectivity of 950, which is ingood agreement with the literature reported selectivity of 1200.Likewise, ligand 2, based on the same parent amino acid sequence as 1,was found to have high 1R/4R selectivity; however, its 1R/5R selectivitywas substantially lower. Unfortunately, ligands 3 and 5-8 were found tobe not at all selective for MC1R, with ligand 3 showing no affinity forany of the receptors tested. Results for ligands 5 and 7 deviate fromthat which has been previously reported; this discrepancy may be due todifferences in the binding assays used to derive the affinity constants.The K_(i) values are based on europium time-resolved fluorescenceassays; however, previously determined EC50 values for these ligandswere derived via ¹²⁵I-labeled competitive binding assays.

As 1 was determined to be the ligand with the highest hMC1R affinity andselectivity, it was chosen for modification with a terminal alkyne forattachment of a triblock polymer micelle. Compound 4 did not demonstratea loss of affinity of MC1R following alkyne functionalization. This isin good agreement with the literature data for MC1R specific ligands,which argues that functionalization at the N-terminus of the peptidedoes not negatively impact binding due to the location of this region ina large hydrophobic cavity or open extracellular part of the receptor,rather than in a confined pocket[20, 28, 29].

As predicted, 1 and 2 have similar binding profiles given the similarityin their structures; however, it was surprising to see a complete lossof affinity in 3. The differences in affinity among these three ligandsarise from the structural differences at the N-terminal end of thepeptide, given that they all share the same R—HfRW—NH₂ parent scaffold.However, whereas 1 and 2 contain Ph-(CH₂)₃—CO— and Ac-Hpe groups, bothof which are non-polar, at the N-terminus, 3 contains a4-hydroxyPh-CH═CH—CO—, which is more polar due the incorporation of thehydroxyl. Conversely, several analogues of 3 possess low nanomolaraffinities against MC1R with varying selectivities (Table 3). Thus, itseems reasonable to conclude that the loss in affinity experienced by 3results from the incorporation of the alkene, rather than the increasedpolarity from the addition of the hydroxyl group. While the exactreasons behind the affinity of 3, or lack thereof, remain unclear, it isplausible that incorporation of the alkene in this ligand causes thepeptide to adopt too rigid a structure, thereby reducing its ability toconform to the receptor binding pocket.

Ligands 5-8 are about twice as large and display binding affinitiesone-to-two orders of magnitude higher with hMC1R as compared to ligands1-4. Ligands 6 and 8 were synthesized as analogues of ligands 5 and 7,respectively, to be used for potential attachment to nanoparticles. Thesimilarity in binding affinities of 5 versus 6 and 7 versus 8 furtherdemonstrates that the H-terminal end of these peptides is a suitablelocation for the placement of an attachment of a scaffold, as it doesnot seem to impact the binding ability of the ligand.

A targeted triblock polymer micelle was prepared by combining 10%4-targeted polymer with 90% untargeted polymer. As a control,competitive binding assays were performed with targeted free polymer anduntargeted micelles. As previously stated, the 4-targeted micelleexhibits an increased binding avidity to the hMC1R receptor as comparedto the free polymer and a slightly weaker avidity than the nativeligand. The increase in binding avidity for the targeted micelles ascompared to the targeted free polymer is noteworthy in that it (1)demonstrates the in vitro stability of the micelle; and (2) it indicatesthat the binding avidity of the 4-targeted micelles may be benefitingfrom multivalent interactions.

While it may be tempting to expect the presence of multivalentinteractions to have a more profound effect on the binding affinity ofthe micelle system, it is important to remember that multivalency is acomplex thermodynamic issue that is influenced by a number of variables,including the enthalpies for each ligand upon individual binding eventsand the entropic consequences experienced by the micelle polymer uponbinding. Additionally, ligand proximity and steric repulsions betweenligands and polymer chains have been shown to be important factors thatinfluence the degree, if any, to which multivalency is experienced inmicellar systems[35]. It is also important to note that improved avidityobserved through multivalent interactions are most typically seen whileusing ligands with relatively low affinity. In the case of 4, it isunlikely that multivalent interactions would greatly enhance bindingavidity given the high affinity of the targeting ligand for binding theMC1R receptor[35, 36]. Finally, the experimental set-up also is afactor. For example, each polymer is composed of roughly 100 polymerchains, meaning that at 10% targeting, roughly 10 targeting groups arepresent per micelle. While this may initially seem like a high number,it is essential to consider the accessibility of these ligands to cellsurface, as the experimental set-up of the time-resolved fluorescencebinding assay does not necessarily lend itself conducive to multivalentinteractions in three dimensions. As a consequence of the assay design,cells are adhered to plates in a monolayer and are only exposed to thetargeted particles on one surface. This limited exposure, combined withthe inflexible nature of the micelle, arguably can explain the slightdecrease in binding affinity experienced in these supramolecularsystems.

What is surprising and counter-intuitive is the slight decrease inbinding affinity experienced between the targeted polymer, targetedmicelles and the native ligand. However, this decrease in affinity maybe the result of the conjugation of a large, flexible PEG group to arather small ligand. In addition to adding to the entropy of the ligandsystem through increased flexibility and size, PEG chains are known tohave at least moderate interaction with non-polar hydrophobicgroups[37]. Consequently, it is possible that the PEG moiety on the endof the triblock polymer is weakly interacting with the hydrophobic aminoacids of the targeting group, thereby decreasing its affinity for MC1R.

An hMC1R ligand was identified in literature and modified by theinventors for attachment to a triblock polymer micelle.Functionalization and subsequent attachment of the ligand to a 100 nmpolymer micelle resulted in a slight decrease in affinity to MC1R.Presumably, this decrease results from the thermodynamic hurdlesencountered in appending a small peptide to a large nanoparticle, aswell as an inherent handicap in the assay design. As mentioned in theintroduction, three hurdles must be overcome in the design of aneffective targeted nanoparticle delivery system: (1) it must be insuredthat there is no loss of ligand affinity resulting from the attachmentof a small peptide to a large nanoparticles, or any such loss inaffinity must be compensated by multivalent binding interactions; (2)nanoparticles must be inherently stable; and (3) nanoparticles must besufficiently small to escape the vasculature and enter the tumor. Theinventors believe that the invention addresses the first two of theseconcerns. The ligand remains selective after attachment and theincreased binding affinity observed between the free 4-targeted polymerand 4-targeted micelle have demonstrated the in vitro stability of thesystem. Additionally, based on DLS data, the inventors are confidentthat the micelles are of sufficient size to escape the vasculature andin vivo studies to evaluate the selectivity and stability of thistargeted micellar system in mice are currently underway.

MC1R-ligand 1 is known to have stronger affinity and higher selectivityto hMC1R than NDP-α-MSH. MC1R-ligand 4 conjugated with the triblockpolymer to form a stable micelle upon crosslinking or to formuncrosslinked micelles of 70-100 nm loaded with Gd-Texaphyrin, aT1-weighted MR radiation enhancer shown below, at 0.05 to 10% weight.FIGS. 5A and B are plots of competitive binding of the MC1R-ligand 4conjugated with the triblock polymer, referred to in the Figure asML21-1, which shows that nanomolar binding affinity the micelle retainednanomolar binding affinity and a significantly higher affinity isobserved for the crosslinked micelle over uncrosslinked micelles. Invitro studies displayed measurable contrast for Gd-Tx at lowconcentrations as indicated in FIG. 6. As can be seen in FIG. 7,micelles in row 3 show higher T1 values than those in row 4, even thoughboth contain 0.01 mg/mL Gd-Tx. This indicates that micelles with higherpercentages of encapsulated Gd-Tx within a single micelle, as tabulatedin FIG. 8, are experiencing a T2 weighting as a result of the tightlypacked gadolinium. Micelles with Gd-Tx encapsulated at 5%, 0.5% and0.05% were prepared at 0.1 mg/mL Gd-Tx and measurements were made whereT1 values indicate that micelles with lower Gd-Tx encapsulated producelower T1 values as indicated in Table 4, below.

TABLE 4 T1 Values for Gd-Texaphyrin Containing Capsules [micelle][Gd-Tx] T1 (% Gd-Tx w/w) (mg/mL) (mg/mL) (s) 5 0.2 0.01 2.40 0.5 2 0.012.35 0.05 20 0.01 1.64 H20 2.89

In vivo studies were carried out where mice were injected with micellesat 12 mg/mL Gd-Tx concentration. T1-weighted images were obtainedpre-injection and at 4 hours, 12 hours, 24 hours and 48 hours afterinjection, for Gd-Tx containing micelles that contained the MC1R-ligandfor targeting (T) or lacked the ligand (UT) and were either crosslinked(XL) of not (UXL), as shown in FIG. 9. No contrast was visible at 4hours and the contrast begins to clear at 48 hours. Only mice injectedwith MC1R-ligand comprising crosslinked micelles (T, XL) exhibitedappreciable contrast, indicating that only these MC1R-ligand comprisingcrosslinked micelles were sufficiently stable to enter and persist inthe tumor. FIG. 10 shows the enhancement of the tumor image by thepresence of the MC1R-ligand comprising crosslinked micelles. Further invivo MRI results are shown in FIGS. 18-22.

The MT-XL micelles are successful at specifically delivering atheragnostic agent to the site of the tumor. Previous systems havefallen short of this goal in that they have encountered wide-spreadtoxicity, especially in the liver, kidneys and spleen. The inventorshave successfully demonstrated that MT-XL micelles: (1) are specific fortheir target; (2) are able to deeply permeate the tumor; and (3) show along tumor half-life, making them ideal for therapy. While this examplefocuses mainly on tumor imaging, similar constructs can be used tospecifically deliver high doses of chemotherapeutic agents, such astaxanes, or other agents, to MC1R-expressing tumors or otherMC1R-expressing cells or tissues, with low systemic toxicity.

REFERENCES

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Materials and Methods for Example 2

Cell Culture.

A375 human malignant melanoma cells were grown in Dulbecco's ModifiedEagle's Medium (DMEM) containing 10% fetal bovine serum (LifeTechnologies, Gaithersburg, Md.), 100 units/mL penicillin and 100 μg/mLstreptomycin in 5% CO₂ at 37° C. The cell line was obtained fromAmerican Type Culture Collection (ATCC), expanded for two passages, andcryopreserved. All experiments were performed with cells of passagenumber less than 25. Cells were authenticated a negative for mycoplasmaby testing at the ATCC and were monitored by microscopy and confirmed tomaintain morphological traits over subsequent passages.

DNA Microarray Analysis of Melanoma Cell Lines.

RNA extracts of melanoma cell lines were analyzed using the AffymetrixU133A array platform (33). Cell lines represented on the array are: 5melanocyte lines—FOM101.1, FOM103.1, FOM104.1, FOM113.1, FOM99.1; 10primary tumor lines—SbC12, WM1366, WM1361A, WM793, WM1819, WM278,WM3248, WM35, WM75, WM983A; 11 metastatic lines—WM1321, WM1346, WM1361B,WM858, WM1617, WM164, WM1727A, WM239A, WM46, WM51, 1205Lu; and 2 linesof unknown stage-origin/phenotype—WM3451 and WM1799. Data generated fromthese arrays have been published previously (33, 34) and have beendeposited in the NCBIs Gene Expression Omnibus (GEO,http://www.ncbi.nlm.nih.gov/geo/). Data are accessible using GEO Seriesaccession GSE4845.

DNA Microarray Analysis of Patient Samples.

Affymetrix expression data for MC1R in patient tissue samples werecompiled from publicly available datasets. The CEL files for four of thenormal skin samples and the skin tumor samples were downloaded from theGEO database (http://www.ncbi.nlm.nih.gov/projects/geo/index.cgi), dataseries GSE7553. Normal tissue data, including additional normal skinsamples were from the GEO data series GSE7307, Human Body Index. The CELfiles were processed using the MAS 5.0 algorithm (Affymetrix, SantaClara, Calif.) and screened through a rigorous quality control panel toremove samples with a low percentage of probesets called present by theMAS 5 algorithm, indicating problems with the amplification process orpoor sample quality; high scaling factors, indicating poor transcriptabundance during hybridization; and poor 3′/5′ ratios, indicating RNAdegradation either prior to or during processing. The remaining sampleswere normalized to the trimmed average of 500 in the MAS 5 algorithmbefore comparison of the expression values across tumors and normalsamples.

Immunohistochemistry (IHC) of Melanoma Tissue Microarray (TMA).

A TMA was constructed at the Moffitt Tissue Core containing human tissuesamples of formalin-fixed and paraffin-embedded (FFPE) specimens. TheTMA contains the following samples 11 normal skin tissue, 11 normal skinfrom melanoma, 10 compound nevi, 10 junctional nevi, 10 intradermalnevi, 40 Clark's atypical dysplastic nevi, 15 primary cutaneous melanomain situ, 15 primary cutaneous melanoma (0.1-0.75 mm), 15 primarycutaneous melanoma (0.75-1 mm), 15 primary cutaneous melanoma (1-2 mm),15 primary cutaneous melanoma (2-4 mm), 15 primary cutaneous melanoma(>4 mm), 10 melanoma distant metastasis-M1, 10 melanoma distantmetastasis-M2, 10 melanoma distant metastasis-M3, 40 melanoma inregional lymph nodes, 15 primary mucosal melanoma. The TMA consists ofcylindrical punches of the FFPE blocks using a Manual Tissue Arrayer(Beecher Instruments, Sun Prairie, Wis.). The same method was previouslyreported for construction of a Ewing sarcoma TMA at the Moffitt TissueCore Facility (35), except the melanoma TMA has only one sample per case(duplicate samples in Ewing) due to the large number of cases. Noidentifiable human subject information was associated with the melanomaTMA.

Rabbit MC1R polyclonal antibody, 1:200 dilution, (GTX70735, GeneTex) wasused for staining with diaminobenzidine (DAB). The slides were scannedin the Moffitt Analytical Microscopy Core Facility (AMC) using an AperioScanScope XT digital slide scanner (Aperio, Calif.). The digital imageof each sample was evaluated by the study pathologist (TWM). Scoringranged from 0 to 9 and was derived from the product of stainingintensity (0-3+)×the percentage of positive tumor (on a scale of 0-3).Scores ≥4 are considered moderate to strong positive.

Generation of Stably Transfected A375 Cells Bearing the MC1R Gene.

pCMV6 containing Homo sapiens MC1R and neomycin as a selection markerwas purchased (Origene, Rockville, Md.). To identify the optimalconcentration for selection, a range (100-1000 μg/ml) of G418(Invitrogen) was tested on cells. A375 cells were transfected with 5 ugof the vector. In response to G418, massive cell death was observedafter ˜5 days. After 2 weeks, resistant colonies appeared. Largecolonies were selected and transferred to individual plates. The clonewith the highest expression of MC1R was determined using qRT-PCR aspreviously described (36). MC1R specific primer sets were designed usingGene Runner Software for Windows version 3.05: forward,5′-AATGTCATTGACGTGATCACCTG-3′ (SEQ ID NO:12) and reverse,5′-GCAGTGCGTAGAAGATGGAGAT-3′ (SEQ ID NO:13). β-actin was used fornormalization (36). A clone with the highest expression was selected andmaintained in medium containing 300 m/ml of G418.

Characterization of hMC1R Expression on A375 Cells byImmunocytochemistry (ICC).

To verify the cell surface expression of hMC1R, two sets of A375(parental) and A375/hMC1R cells were plated at a cell density of 1×10⁴cells/well on glass coverslips placed at the bottom of culture wells andincubated for 16 hours. Cells were then treated with 30 μg/ml MC1Rantibody (Almone Labs) and 5.0 μg/mL of WGA (Invitrogen) at 4° C. for 10minutes, washed 3 times with PBS, fixed with cold methanol:acetone (1:1)and air dried for 20 minutes. Plates were washed 3 times with warm PBSand incubated with 1:2000 secondary antibody (Alexa-Fluor 680 goatanti-mouse IgG, Invitrogen). After three washes with PBS, coverslipswere mounted with mounting medium and DAPI (Vector Laboratories, Inc.,Burlingame, Calif.). Samples were viewed in the Moffitt AnalyticalMicroscopy Core Facility using an automated Zeiss Observer Z.1 invertedmicroscope using 40×/1.3NA oil immersion objectives through narrowbandpass DAPI and FITC/A488 Chroma filter cubes and NomarskiDifferential Interference Contrast polarizing and analyzing prisms.Images were produced using the AxioCam MRm CCD camera and Axiovisionversion 4.6 software suite (Carl Zeiss Inc., Germany).

Synthesis, Purification and Characterization

Materials.

N^(α)-Fmoc protected amino acids, HBTU, and HOBt were purchased fromSynPep (Dublin, Calif.) or from Novabiochem (San Diego, Calif.). Rinkamide Tentagel S resin was acquired from Rapp Polymere (Tubingen,Germany). The following side chain protecting groups were used for theamino acids: Arg(N^(g)-Pbf); His(N^(im)-Trt); Trp(N^(in)-Trt);Lys(N^(e)-Aloc).

IR800CW maleimide dye was purchase from Licor (Lincoln, Nebr.). Cy5maleimide dye was purchase from Licor (Lincoln, Nebr.). Peptidesynthesis solvents, dry solvents, and solvents for HPLC (reagent grade),and 4-phenylbutyric acid, were acquired from VWR (West Chester, Pa.) orSigma-Aldrich (Milwaukee, Wis.), and were used without furtherpurification unless otherwise noted. Compounds were manually assembledusing 5 to 50 mL plastic syringe reactors equipped with a frit, andDomino manual synthesizer obtained from Torviq (Niles, Mich.). The C-18Sep-Pak™ Vac RC cartdridges for solid phase extraction were purchasedfrom Waters (Milford, Mass.).

Peptide Synthesis.

Ligands were synthesized on Tentagel Rink amide resin (initial loading:0.2 mmol/g) using N^(α)-Fmoc protecting groups and a standard DIC/HOBtor HBTU/HOBt activation strategy. The resin was swollen in THF for anhour, washed with DMF, and Fmoc protecting group removed with 20%piperidine in DMF (2 min+20 min). The resin was washed with DMF, DCM,0.2 M HOBt in DMF, and finally with DMF and the first amino acid coupledusing pre-activated 0.3 M HOBt ester in DMF (3 eq. of N^(α)-Fmoc aminoacid, 3 eq. of HOBt and 6 eq. of DIC). An on-resin test usingBromophenol Blue was used for qualitative and continuous monitoring ofreaction progress. To avoid deletion sequences and slower coupling ratein longer sequences, the double coupling was performed at all steps with3 eq. of amino acid, 3 eq. of HBTU and 6 eq. of DIEA in DMF. Anyunreacted NH₂ groups on the resin thereafter were capped using an excessof 50% acetic anhydride in pyridine for 5 min. When the couplingreaction was finished, the resin was washed with DMF, and the sameprocedure was repeated for the next amino acid until all residues werecoupled.

Aloc Cleavage.

The orthogonal protecting Aloc group of C-terminal Lys was cleaved asfollows. The resin was washed with DCM then flushed with argon for 10min. A cleavage mixture of dimethylbarbituric acid (5 equiv.), Pd(TPP)₄(0.2 equiv.) in DCM (0.5 M solution) was flushed with argon and injectedinto the syringe. The reaction mixture was stirred for 30 min thenrepeated. The resin was washed with DMF, 10% DIEA in DMF, DMF, 2% sodiumdiethyldithiocarbamate trihydride, 10% DIEA in DMF, DCM and DMF. TheTrt-Mpr was attached to the free amine via HBTU coupling as describedabove.

Cleavage of Ligand from the Resin.

A cleavage cocktail (10 mL per 1 g of resin) of TFA (91%), water (3%),triisopropylsilane (3%), and 1,2-ethylenedithiol (3%) was injected intothe resin and stirred for 4 h at room temperature. The crude ligand wasisolated from the resin by filtration, the filtrate was reduced to lowvolume by evaporation using a stream of nitrogen, and the ligand wasprecipitated in ice-cold diethyl ether, washed several times with ether,dried, dissolved in water and lyophilized to give off-white solidpowders that were stored at −20° C. until purified. The crude compoundwas purified by size-exclusion chromatography.

Labeling Procedure.

The purified thiol compound (1 mmol) was dissolved in 1 mL DMF andreacted with 1 equiv. of IR800CW or Cy5 maleimide under argonatmosphere. The reaction was monitored by HPLC and additional aliquotes(0.1 equiv.) of dye were added until the reaction complete. The compoundwas purified by HPLC.

Purification and Analysis.

Purity of the peptides was ensured using analytical HPLC (WatersAlliance 2695 separation model with a dual wavelength detector Waters2487) with a reverse-phase column (Waters Symmetry, 3.0 75 mm, 3.5 μm;flow rate=0.3 mL/min). (Conditions: HPLC, linear gradient from 10 to 90%B over 30 min, where A is 0.1% TFA and B is acetonitrile). Sizeexclusion chromatography was performed on a borosilicate glass column(2.6×250 mm, Sigma, St. Louis, Mo.) filled with medium sized SephadexG-25 or G-10. The compounds were eluted with an isocratic flow of 1.0 Maqueous acetic acid. Solid-Phase Extraction (SPE) was employed wheresimple isolation of final compound was needed from excess salts andbuffers for e.g., lanthaligand synthesis. For this purpose, C-18Sep-Pak™ cartridges (100 mg or 500 mg) were used and pre-conditionedinitially with 5 column volumes (5 times the volume of packed columnbed) each of acetonitrile, methanol, and water, in that order. Afterloading the compound, the column was washed several times with water,and then gradually with 5, 10, 20, 30, 50, and 70% of aqueousacetonitrile to elute the peptide. Structures were characterized by ESI(Finnigan, Thermoquest LCQ ion trap instrument), MALDI-TOF or FT-ICRmass spectrometry. An appropriate mixture of standard peptides was usedfor internal calibrations.

Binding Assays.

A375 melanoma cells engineered to express MC1R were used to assessligand binding in a competitive binding assay as described before (32).

The receptor number of A375/MC1R (engineered cells to express MC1R), wasdetermined using saturation binding assay following a previouslydescribed method (37), except that Eu-DTPA labeled NDP-α-MSH was used asa test ligand and 5 μM of NDP-α-MSH was used as a blocking ligand. Thesecells were used as a MC1R high expressing line, while the parental(A375) cells were used as a low expressing line with 400±93 sites/cell(38).

In Vitro MC1R Probe Uptake Study.

To study the uptake of the MC1R probe in vitro, two sets of A375(parental) and A375/hMC1R cells were plated at a cell density of 1×10⁴cells/well on glass coverslips placed at the bottom of culture wells andincubated for 16 hours. Cells were incubated in media containing 15 nMprobe and uptake evaluated by fluorescence microscopy at different timepoints from 40 seconds to 15 minutes. To determine specificity, MC1Rreceptors were blocked by a 10 minute pre-incubation with 2 μM ofNDP-α-MSH prior to addition of MC1R probe and images acquired 1 minuteafter addition of labeled probe. Samples were viewed using an AxioObserver Z1 inverted fluorescence microscope (Carl Zeiss, Inc, Germany)using 40×/1.3NA oil immersion objectives through a narrow band Cy5filter. Cy5 fluorescence images were prepared with a DIC overlay imageusing Axiovision 4.6 software (Carl Zeiss, Inc, Germany).

In Vivo Uptake Study: Intravital Imaging of the Dorsal Skin-Fold WindowChamber Tumor Xenograft Model.

All procedures were carried out in compliance with the Guide for theCare and Use of Laboratory Animal Resources (1996), National ResearchCouncil, and approved by the Institutional Animal Care and UseCommittee, University of South Florida. A dorsal skin-fold windowchamber was used to study the pharmacokinetics of imaging agent uptakeimmediately after i.v. injection of the probe. Starting 3 days prior tothe surgery, mice were daily administered 1 ml of sterile salinesubcutaneously at the planned site of the window in order to loosenconnective tissue and create a receiving “pocket”. SCID mice wereprepared immediately before surgery; the surgical site was shaved, andthen a combination of oxygen and isoflurane was used for anesthesia(animals underwent induction with 3.5% isoflurane and were maintained at1.5-2.0%). The window chamber method utilizes a titanium steel orplexiglass “saddle” that is sutured to the back of the mouse and holds aflap of dorsal skin vertically away from the mouse's body. A small“window” of skin (approximately 5 mm in diameter) was surgically excisedfrom the retained skin flap of the anesthetized SCID mouse. Tumorconstructs were engineered using the tumor droplet method. A375/MC1R orA375 parental melanoma cells were suspended in 2.5 mg/ml of type Icollagen (BD Biosciences #354249) and 1×DMEM at a final concentration of2.5×10⁶ cells/ml. Using a 48-well non-tissue culture plate, a 15 μl dropof the tumor cell suspension was polymerized in the center of the well.After brief polymerization (10-15 minutes) at 37° C., a microvesselouter layer was added that completely surrounded the droplet. Themicrovessel outer layer consisted of 3 mg/ml of type I collagen, 1×DMEMand, 12,000 to 15,000 GFP expressing rat microvessel fragments/ml. After4-6 days of culture at 37 C, the constructs were then implanted into the“window chamber” by placing them directly onto the exposed subcutaneousfascia. A glass cover was then attached to the center of the “saddle” tocover the fascia and implanted tumor cells. Post-operatively, tumorgrowth and micro-circulation can be visualized in this model forapproximately a month.

GFP expressing microvessel fragments were prepared from the epidydimalfat of transgenic GFP SpragueDawley rats. A rat was anesthetized, andthe abdomen sprayed with 70% ethanol. Skin was clamped just below peniswith a hemostat and an incision made with large scissors starting in thecenter and cutting laterally. A small incision was made into the scrotumusing small scissors exposing the epidydimal fat. The epidydimal fatpads were carefully removed using forceps and the animal euthanized byextending the incision and performing a thoracotomy while remainingunder deep anesthesia. Microvessels were then prepared as follows:Epididymal fat pads were removed and digested with collagenase (2mg/ml). Following digestion, large tissue debris was removed using a 500μm filter and subsequent filtration step done with a 30 μm filter tocollect the microvessel fragments. Microvessel fragments were then addedto a solution of type I rat tail collagen at a final concentration of 3mg/ml (BD Bioscience, San Jose, Calif.) with 1×DMEM, and kept on ice tokeep collagen from polymerizing. This preparation was added to thecancer cell droplet and placed under the glass cover of a dorsalskin-fold window chamber on a SCID mouse following a 3 to 4 day in vitroincubation. As the tumor xenograft is established, rat microvesselsbecome patent with the mouse vasculature (39).

Seven days after implantation of microvessels and tumor cells, mice wereintravenously injected with 100 μl of 5% 10,000 MW Cascade Blue Dextran(Invitrogen, CA) in sterile H2O to verify microvessel patency. Then, 5nmol/kg of the MC1R-Cy5 probe was injected into the tail vein. Confocalimages of probe uptake into the melanoma tumor cells were continuouslyacquired prior to, during and after injection of probe using the OlympusFV1000 (MPE) Multiphoton Laser Scanning Microscope (Lisa Muma WeitzAdvanced Microscopy and Cell Imaging facility at USF) using a 1.25× and25× lens and acquisition rate of 3570 Pixels/minute. The Cy5 conjugatedligand was measured by exciting the ligand with an IR laser at 635 nmand the emitted light was detected using a 655-755 nm filter.

Tumor Xenograft Studies and Fluorescence Imaging.

Female nu/nu mice 6-8 weeks old (Harlan Sprague Dawley, Inc.,Indianapolis, Ind.) were injected subcutaneously (s.c.) with 1×10⁶ MC1Rexpressing A375 cells in the right and the parental one in the leftflank. Tumor volume was determined with calipers using the formula:volume=(length×width²)/2. Once tumors reached 500-800 mm³, 1 nmol/kg-30nmol/kg of MC1R imaging probe in 100 μL sterile saline was injected intothe tail vein. In vivo fluorescence images were acquired using theOptix-MX3 (Advance Research Technologies, Inc. a subsidiary of SoftScanHealthcare Group, Montreal, Canada). Animals were positioned on aheating pad and anesthetized using isoflurane (flow 2-2.5 l/min).Fluorescence images were acquired using a scan resolution of 1.5 mm anda 790-nm pulsed laser diode with 40 MHz frequency and 12-ns time window.Images were analyzed using Optix-MX3 Optiview Software (version 3.01).Autofluorescence background was subtracted by determining the mean tumorfluorescence signal prior to injection, then mean normalized intensityvalues were obtained within a ROI on these images.

Ex Vivo Studies.

Tumors were excised and center slice of each tumor was imaged using bothOptix-MX3 (as described above) and IVIS 200 imaging system (CaliperLifeSciences, MA). For IVIS 200 acquisitions, the standard ICGexcitation and emission filter set was used for imaging. After imaging,the slices were fixed in formalin and embedded in paraffin forhistology. Formalin fixed sections (5 μm) were stained with hematoxylinand eosin (H&E). Sections were also IHC stained with MC1R primaryantibody as described above for TMAs.

Biodistribution Studies.

Mice were imaged and euthanized at 2-72 hr post-injection. Tumors,kidneys and liver were excised, rinsed with PBS, blotted dry, and thenimaged ex vivo with Optix-MX3 as described above. Images were analyzedas described above.

Log D.

The log of the octanol-water partition coefficient at pH 7.4 (logD_(7.4)) was determined by miniaturised shake flask assay. Briefly, (200μL to 1 mL) n-octanol (Sigam) was added to a solution of the testcompound prepared in PBS (25 mM NaH₂PO₄/Na₂HPO₄ buffer, pH 7.4, Sigma,HPLC grade). Then, three different ratios of octanol to PBS buffer wereprepared. The mixture was stirred in a vortex mixer at room temperaturefor 1 min and then two layers were separated by centrifuge. Theconcentration of compound in each layer was determined by HPLC(Poroshell 120 EC-C18 column) using (26% acetonitrile in water, 0.1%TFA) in 280 nm channel. All sample injections were performed 3 times,and the results were averaged to yield the final values.

Statistics.

Data are represented as mean±s.d. All statistical analyses wereperformed with GraphPad Prism version 5.01. Unpaired Student's t-testwas used to determine the statistical significance of differencesbetween two independent groups of variables. For all tests, a p≤0.05 wasconsidered significant.

Example 2—Development of a Melanoma Targeted Probe for Imaging ofMelanocortin Receptor 1 (MC1R)

Nodal metastases are frequently the initial manifestation of metastaticspread in patients with melanoma and accurate determination of nodalstatus is important for both prognostic evaluation and treatmentplanning. The melanocortin 1 receptor (MC1R) is overexpressed in mosthuman melanoma metastases, thus making it a promising target for imagingand therapy of melanomas. In this study, using DNA and tissuemicroarray, MC1R expression was analyzed in different normal tissues andmelanoma samples, confirming the expression of MC1R in a large fractionof patients with melanoma. The inventors had developed a peptidomimeticligand with high specificity and affinity for MC1R. Here, the inventorshave conjugated this ligand to a near-infrared fluorescent dye togenerate a MC1R specific optical probe (MC1RL-800, 0.4±0.1 nM K_(i)).The uptake of the probe was studied in engineered A375/MC1R cells invitro as well as in vivo by intravital fluorescence imaging, showinginternalization of the probe. The in vivo tumor targeting of MC1RL-800was evaluated by intravenous injection of probe into nude mice bearingbilateral subcutaneous tumors of A375 cells with low MC1R receptornumbers and engineered A375/MC1R cells. Fluorescence imaging showed thatthe agent has higher uptake values in tumors with high expressioncompared to low (P<0.05), demonstrating the effect of expression levelson image contrast-to-noise. In addition, the tumor uptake wassignificantly blocked by co-injection with excess NDP-α-MSH peptide(P<0.05), indicating specificity of the probe in vivo. Thebiodistribution of MC1RL-800 was investigated in xenograft bearing mice,showing high kidney uptake as early as 30 min post-injection. As kidneyis known to express the melanocortin receptor family member (MC4R andMC5R), kidney uptake of the probe was reduced significantly (P<0.05) byco-injection of a ligand the inventors have previously identified tohave higher MC5R affinity compared to MC1R. The pharmacokinetics ofprobe uptake and clearance was also characterized using athree-compartment mathematical model. The MC1R-specific imaging probedeveloped in this study displays excellent potential for in vivodetection of melanoma metastases.

Malignant melanoma is the most common cause of death from cutaneousmalignancies and the fastest increasing cancer in the U.S. (1, 2).Assessment of metastatic spread to lymph nodes draining the tumor siteis very important not only for staging and prognosis but also foradjuvant therapy in melanoma (3-5). Sentinel lymph node biopsy (SLNB) isthe current gold standard for evaluating regional lymph node involvement(6). The sentinel lymph node (SLN) receives lymph draining directly fromthe tumor site. In this method, a radio colloid is injectedintradermally around the tumor and SLNs are determined, removed andexamined by histological methods for detection of intranodal metastasis(7). If cancerous, the patient is then offered a completionlymphadenectomy to remove the remainder of the lymph nodes in thatanatomic area. This method has limitations, as it is a surgicalintervention with all the ensuing complications from infection tolymphedema (8), and 80% of patients are negative for lymph nodemetastasis and therefore have undergone an unnecessary procedure (9).

Standard imaging techniques have been used for assessment of regionalnodal status, including CT, ultrasound, MRI, and ¹⁸F-FDG PET (10, 11).However, each of these techniques have limitations as tools fordetection of metastatic melanoma. For example, ultrasound is limited todetection of superficial nodes. ¹⁸F-FDG PET lacks sufficient sensitivityto detect micrometastasis in regional nodes, especially for the initialassessment of early-stage melanoma metastasis where tumor volume issmall or metabolically inactive (12-14). In addition, this tracer cannot discriminate between malignancy and inflammatory lymphadenopathy(15, 16). None of these imaging modalities are specific for enhancementof melanoma metastases. Therefore, melanoma-specific molecular imagingprobes are needed for the non-invasive detection of metastasis with highsensitivity for staging of regional lymph node involvement, and fordetection of distal metastases for diagnosis and to follow therapyresponse. There is also increasing interest in the use of novel targetedtherapies in malignant melanomas that are resistant to most systemictherapies (17).

Melanoma progression is associated with altered expression of cellsurface proteins, including adhesion proteins and receptors (18-20). Ithas been estimated that over 80% of malignant melanomas express highlevels of the MC1R (21). MC1R is a member of a family of five Gprotein-coupled receptors (MC1R-MC5R) for melanocortins (22-24), such asmelanocyte stimulating hormones (MSH). Because of the high expression ofMC1R in melanoma, it has been investigated as a target for selectiveimaging and therapeutic agents and a number of selective ligands havebeen developed (25-27). One of these ligands, [Nle⁴,D-Phe⁷]-α-MSH(NDP-α-MSH) has a high affinity against MCR, except MC2R, and has beeninvestigated extensively (28-31).

In this study, the inventors evaluated the expression of MC1R throughimmunohistochemistry (IHC) and DNA microarray analysis in both melanomapatient samples and cell lines. While MC1R is not a novel target, thisrepresents the most extensive study on its distribution in melanoma todate. In addition, the inventors recently described a high affinityselective ligand against MC1R with lower affinity for MC4R or MC5R (32).By conjugating a near-infrared fluorescent dye to this ligand, we havedeveloped a MC1R specific molecular imaging probe (MC1RL-800). Theinventors have used this probe to image the expression of MC1R in vivofollowing intravenous injection into nude mice bearing bilateral high-and low-MC1R expressing tumors. In vivo tumor cell uptake of this probewas studied by intravital imaging of a dorsal skin-fold window-chambermouse xenograft tumor model. The in vivo biodistribution andpharmacokinetics of the probe was also studied. The molecular imagingprobe designed in this study has potential for the detection and stagingof melanoma metastases that overexpress the MC1R, and could be used forthe targeted delivery of therapy.

Results

A. MC1R Expression in Patient Tissue Samples

It has been estimated that 80% of malignant melanomas express highlevels of MC1R (21). For further confirmation and to characterize mRNAexpression in patient tissue samples, the inventors analyzed publiclyavailable DNA microarray data sets, which showed that MC1R mRNAexpression was highly and generally expressed in a large fraction ofmelanomas (FIG. 24A, note log scale). In contrast, MC1R expression wasnot elevated in other skin cancers, normal skin and organs involved intoxicity and drug clearance, i.e, heart, lung, spleen, liver and kidney.

To determine MC1R protein expression in patient samples,immunohistochemistry (IHC) was performed on a melanoma tissue microarraycontaining 267 samples. FIG. 24C shows representative staining in normalskin relative to staining in a primary cutaneous melanoma, distantmetastasis and lymph node metastasis. None of the normal skin samples(n=19) had staining with a pathology score of ≥4, i.e. homogeneousmoderate to high staining (Table 5). Benign lesions (n=65) and samplesof local invasion to regional lymph nodes (n=35) had percentages of ≥4staining ranging from 15 to 33%. A relationship between primarycutaneous melanoma lesion size and pathology score was observed, withsmaller lesions ranging from melanoma in situ to lesions 1 mm indiameter scoring 18% to 33%≥4, and lesions from 1 mm to ≥4 mm indiameter scoring 46 to 79%≥4. Primary mucosal melanomas (n=11) andmelanoma from distant metastasis had scores ranging from 40% to 67%≥4.

TABLE 5 IHC Scoring of MC1R Expression in Patient Tissue SamplesPathology score Tissue type n 0 1 2 3 4 6 9 % = 4 normal skin 19 0 1 513 0 0 0 0 compound nevi 9 0 0 0 6 0 3 0 33 junctional nevi 5 0 0 0 4 01 0 20 intradermal nevi 7 0 0 0 5 0 2 0 29 Clark's, atypical, dysplasticnevi 33 0 1 5 22 0 5 0 15 Primary cutaneous melanoma (in situ) 11 0 0 09 0 2 0 18 Primary cutaneous melanoma (0.1-0.75 mm) 12 0 2 1 5 0 4 0 33Primary cutaneous melanoma (0.75-1 mm) 13 0 0 3 7 0 3 0 23 Primarycutaneous melanoma (1-2 mm) 13 0 0 3 4 0 6 0 46 Primary cutaneousmelanoma (2-4 mm) 12 0 0 1 3 1 6 1 67 Primary cutaneous melanoma (>4 mm)14 0 1 1 1 0 8 3 79 Primary mucosal melanoma 11 0 1 1 3 0 4 2 55Melanoma in regional lymph nodes 35 1 3 10 12 0 9 0 26 Melanoma distantmetastasis, M1 9 0 1 0 4 0 3 1 44 Melanoma distant metastasis, M2 9 0 01 2 0 5 1 67 Melanoma distant metastasis, M3 10 0 1 0 5 0 4 0 40B. MC1R Expression in Melanoma Cell Lines

MC1R expression was examined on a previously published DNA microarray ofa panel of melanoma cell lines (33, 34) (FIG. 24B). Moderate to highexpression was observed in all primary melanocytes and NRAS-positivemelanoma cells had low to moderate expression. In contrast, expressionin BRAF-positive melanoma cells was highly heterogeneous, with somelines exhibiting very low expression (WM 858, 7938, 1799, 35) and somewith extremely high expression (WM 164, 1727A, 1819, 239A). Notably, 3of the 4 high expressing lines were metastatic: WM164, WM239A andWM1727A.

C. Characterization of MC1R Expression in A375/MC1R Cells

A375 malignant melanoma cells were transfected to stably overexpressMC1R for evaluation of the MC1RL-800 imaging probe both in vitro and invivo. The A375 malignant melanoma cell line was chosen due to their verylow endogenous expression of MC1R (38, 40), providing both low- andhigh-expressing cells for the in vitro as well as in vivo selectivitystudies. qRT-PCR of A375 and A375/MC1R cells revealed the mRNA geneexpression level in the engineered cells to be 457, compared to a geneexpression level of 4.5 in the parental cells. For further confirmation,hMC1R expression on the cell surface of the cells was characterizedthrough ICC (FIG. 25A). Both engineered A375/hMC1R and parental A375cells were incubated with the nuclear marker DAPI, the plasma membranemarker WGA and an MC1R antibody conjugated to a fluorescent dye (Alexa555). The merged images illustrate colocalization of MC1R (red) withmembrane marker (WGA, green) indicating accumulation of the receptor onthe cell-surface (yellow). Notably, the parental A375 line does appearto have a detectable amount of MC1R antibody binding to the cellssurface.

To determine MC1R receptor number on the cell surface, saturationbinding assays were performed using Eu-NDP-α-MSH. Increasing amounts ofEu-NDP-α-MSH were added to A375/MC1R cells. Non-specific binding wasdetermined in the presence of 5 μm unlabeled NDP-α-MSH (FIG. 25B).Results indicate that the Kd, Bmax and receptor number were 1.8 nM,668,046±67,108 and 75,000 respectively.

D. Synthesis and Characterization of MC1R Targeted Probes

The inventors recently described a high affinity and selective MC1Rligand; 4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂ (SEQID NO:3). K_(i) values for this ligand against MC1R, MC4R and MC5R were0.24 nM, 254 nM and 46 nM, respectively (32). Here, the inventorsfurther describe the attachment of this ligand to a near-infrared (NIR)dye with an excitation at 800 nm(4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(Mpr-IR800CW)-NH₂ (SEQ IDNO:14), LiCor IR800CW Maleimide) as well as Cy5.

To evaluate the binding affinity of the MC1RL-800 and MC1R-Cy5 probes,competition binding assays were preformed on A375/hMC1R cells withEu-NDP-a-MSH as the competed ligand (FIG. 25C). The MC1RL-800 andMC1R-Cy5 probes retained high affinity against MC1R, with an K_(i) of0.4±0.1 nM and 0.3±0.05 nM, respectively, compared to the K_(i) forunlabeled ligand of 0.24 nM Ki (32).

E. Cellular and Tumor Uptake Studies

To study uptake of the MC1R probe onto and into cells, MC1RL-800 probewas incubated with live cells and images were acquired at differenttime-points after incubation using an inverted microscope. The bindingof dye to the surface of A375/MC1R cells was observed as early as 15seconds after incubation and intracellular accumulation was observedwithin 5 minutes (FIG. 26A). No attachment of the probe was observedwhen 2 μM of NDP-α-MSH was used as a blocking agent before adding theprobe to the cells (FIG. 26A).

To study distribution of the probe into tumors and tumor cells,intravital imaging of a dorsal skin-fold window chamber xenograft tumormodel was employed (see Methods). Briefly, the dorsal skin of a mouse isfolded up into a saddle frame, and one side of the skin is removed in acircular region of ˜1 cm in diameter and a round cover slip is placedover the opening, enabling high-resolution microscopic studies (41). Forthis experiment, A375/MC1R cells were mixed with matrigel and GFPexpressing rat microvessels which were xenografted into the windowchamber under the glass cover. Following a 5-7 day period of tumorgrowth, microvessel patency was verified by i.v. injection of blueDextran and regions of the tumor with patent GFP vessels were chosen forstudy (see FIG. 31A). Then, 5 nmol/kg of the MC1R-Cy5 probe wasintravenously injected. Extravasation, tumor cell binding and uptake ofthe probe was observed by continuous confocal microscope acquisitions.FIGS. 31B-1 and 31B-2 show the whole tumor surrounded by GFPmicrovessels at 24 hr after injection of probe using low magnification(1.25 and 4×), while uptake of the probe at different time pointspost-injection are shown in FIG. 26B using higher magnification (25×).The probe was observed to extravasate, penetrate into the tumor and bindto cells as early as 5 min after injection. At five minutes to two hoursafter injection, most of the MC1R probe was observed on the surface ofthe tumor cells, with lesser amounts taken into the cells. At 24 hourspost-injection, the tumor cells had fully internalized the probe.

F. In Vivo Tumor Targeting

To investigate tumor targeting of the probe in vivo, bilateralsubcutaneous xenograft tumors were established with A375/MC1R engineeredcells in the right flank, and A375 parental cells with relatively lowMC1R expression in the left flank. After tumor growth to approximately500-800 mm³, MC1RL-800 was injected intravenously and fluorescenceaccumulation was monitored over time. At 2 h post-injection, the A375tumors with low MC1R expression had significantly lower normalizedfluorescence signal compared to A375/MC1R tumors (P≤0.05, n=3) (FIG.27A-1, left mouse, and FIG. 32A). Representative A357/MC1R tumor slicesin the volume in vivo as well as ex vivo are shown in FIGS. 32B and 32C,indicating heterogeneity of probe labeling within the tumor. The in vivospecificity of the probe was confirmed by co-injection of 0.25 μg ofNDP-α-MSH with 5 nmol/kg of the MC1RL-800 probe. After blocking, theprobe-related fluorescence signal decreased 1.54 fold in the A375/MC1Rtumors (P≤0.05, n=3) and 1.4 fold in A375 tumor (P≤0.05, n=3) relativeto the unblocked tumor at 2 hr after injection (FIG. 27A-1, rightmouse). Ex vivo images of the corresponding center sections of the high-and low-expressing tumors confirmed the in vivo results. IHC stainingconfirmed the high and low MC1R expression in the two tumor types, andareas with the highest IHC staining corresponded to areas with thehighest fluorescence signal (FIG. 27B).

G. Biodistribution and Mathematical Modeling

For biodistribution studies, mice bearing high expressing (A375/MC1R)tumors were injected with probe (n=3), and tissue distribution offluorescence signal determined after removing tumors and organs from 30min to 72 hr post-administration (FIG. 28A). At 30 minutespost-injection, probe was retained at relatively high levels in the MC1Rhigh expressing tumor. At early time points, probe accumulation in thekidneys was significantly higher than in the tumors, e.g at 2 hr afterinjection, kidney signal was 2 fold higher, P<0.001. However, theaccumulation was no longer significant by 72 hr after injection and theprobe was cleared from both tumors and kidneys by 96 hr after injection.MC1RL-800 did not accumulate in the liver and no signal was detected inthe other organs, such as spleen, heart, brain, etc.

The pharmacokinetics of probe uptake and clearance was alsocharacterized using a three-compartment mathematical model, whichincludes tumor, kidney and mouse volumes, and assumes mass conservationof the ligand. This model was used to account for the interference ofthe tumor ligand release in the uptake and clearance dynamics of bloodand kidneys. The results showed, while this effect is negligible inhumans, it is significant in mouse models.

Both ex vivo and in vivo biodistribution studies fit in the model (FIG.28B), suggesting using in vivo fluorescent imaging which provides highsensitivity for biodistribution study instead of sacrificing multiplesanimals at multiple time points. In addition, the probe is cleared atthe same rate from both kidneys and tumor according to the inventors'model.

H. Determination of Log D of MC1RL-800

To measure lipophilicity of the probe, log D (distribution co-efficient)was determined. Lipophilicity is a key determinant of thepharmacokinetic behavior of drugs and can influence distribution intotissues, absorption and the binding characteristics of a drug, as wellas determination of the solubility of a compound (reviewed by (42). Thelog D of MC1RL-800 probe was calculated −2.96, demonstrating highsolubility of the probe but low permeability across the gastrointestinaltract or blood brain barrier.

I. Reduction of Kidney Uptake

Significant renal accumulation was observed as early as 15 min afterintravenous injection of the probe. To determine whether the amount ofthe MC1RL-800 probe injected had an effect on kidney uptake andretention, different amounts of the probe were injected intravenously(i.v.) and the pharmacokinetics of uptake and clearance monitored (FIGS.30A-D). As expected, the lower dose (1 nmol/kg) provided greaterdiscrimination and more rapid renal clearance, compared to the higherdose (5 nmol/kg) (FIGS. 29A-1 and 29A-2). Furthermore, with either dose,the measureable tumor and kidney half-lives were approximately 10 hr.Compared to previous studies, it is possible that this represents thetime for dye to be acid quenched following internalization, and thiswill be characterized more fully in the future using Eu-labeled ligands.

In addition, as the kidneys express MC5R (43, 44) and have little or noexpression of MC1R (vide supra), the inventors hypothesized that renalaccumulation is occurring via off-target binding of the probe to MC5R.To test this, 1 nmol/kg of an MC4R/5R-selective ligand,H-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂ (SEQ IDNO:10), (32) was injected. This agent has Ki values of 5.6, 0.77 and0.71 nM for MC1R, MC4R and MC5R, respectively. Co-injection of MC4R/5Rcompound along with 5 nmol/kg of MC1RL-800 significantly reduced renaluptake, e.g 1.56 fold lower at 2 hr after injection, P<0.05, compared tocontrol animals (FIG. 29B). Whereas the A375/MC1R tumors had asignificantly lower 1.54 fold decrease following blocking. Thus, itappears that the renal uptake of MC1RL-800 is due to off-target binding,which can be pharmacologically blocked.

Early detection of primary melanoma tumors is essential because there isno effective treatment for metastatic melanoma. MC1R is known to beover-expressed in the majority of melanoma patients (21, 45-47),representing one of the few specific targets potentially useful fordiagnosis and therapy of metastatic melanoma. MC1R, has been intensivelystudied as a target for melanoma therapies; however, to the best of theinventors' knowledge, this represents the first publication in which ithas been so extensively evaluated in terms of expression levels inpatient samples and cell lines. In addition, the inventors havedeveloped MC1R specific imaging probes and characterized their tumorcell specificity, binding and uptake both in vitro and in vivo.

It has been previously reported that up to 80% of melanoma cells expresshigh levels of MC1R (21); however, those data were obtained through theuse of radioassays using human cell lines, rather than patient tissuesamples. It is not uncommon for protein expression in cultured celllines to differ from that of primary tumors; in fact, such discrepanciesare common throughout literature and undoubtedly play a role in theincreased sensitivity of cancer lines to chemotherapy relative to solidtumors (48). Previous reports of MC1R expression in human melanoma andhuman uveal melanoma tissues have been as high as 95% (49, 50); however,these studies have taken a “present/not present” binary approach to datascoring. The inventors have rigorously sought to quantify MC1Rexpression in patient samples with the assistance of adermatopathologist. The data that the inventors have collected fromhuman tissues samples suggests that MC1R is moderately to highlyexpressed (with a score of ≥4) on only 40% to 60% of melanoma patienttumors, leading us to believe that earlier reports, citing highexpression of MC1R in 80-95% of melanoma cells (21, 49, 50), may havebeen an over-estimate.

Based on the inventors' analysis of publically available DNA microarraydata from non-affected human tissues, MC1R mRNA expression is notelevated in non-melanoma skin cancer, normal skin cells, or organs whichare typically involved in pharmaceutical clearance, such as the heart,liver, spleen or kidney. These findings are in good agreement withrecent literature (49, 50). Additionally, Salazar-Onfray et.al (50)conducted an analysis of a broad panel of normal tissues through IHC andfound that MC1R was detected only in low levels in adrenal grand,cerebellum and liver, and very weakly in normal appendix, myocardium,kidney and myometrium. Their data indicated that while MC1R is notexpressed at detectable levels on fresh monocytes, in vitro stimulationwith several cytokines such as IL-4, GM-CSF, and IL-10 can induce astrong expression of MC1R. Herein, the inventors report high expressionof MC1R mRNA in melanocytes, and high protein expression in the basallayer of normal skin samples. Melanocytes are known to be present in thebasal layer. The inventors' results indicate that MC1R isheterogeneously expressed in normal skin relative to the ubiquitous andhigh expression observed in a large fraction of melanomas. Theinventors' findings, combined with previous reports from the literature,suggest that MC1R may be useful as a marker for a large subset ofpatients with melanoma for specific targeting.

It has been known that MC1R ligands also show cross-reactivity withother melanocortin receptors, mainly MC4R and MC5R. Since MC4R and MC5Rhave high expression levels in normal tissues including kidney andbrain, MC1R ligands with non-specific binding to MC4R and MC5R are notideal for targeting of melanoma in patients. Recently, the inventorsdescribed a high affinity peptidomimetic ligand against MC1R anddemonstrated very low interaction against MC4R and MC5R, 1000 and 200times lower affinity compared with MC1R, respectively (32). As thisligand had the highest hMC1R affinity and selectivity in the inventors'earlier study, it was chosen for more study here. The use of smallpeptidomimetics as carriers for the delivery of imaging or therapeuticmoieties to diseased tissues offers several advantages such as highbiostability, easy synthesis and modification, faster blood clearance,high-affinity and high-specificity and a low toxicity and immunogenicity(51-55).

In this research, the inventors further developed a molecular imagingprobe by attachment of a near-infrared fluorescent dye IRDye800CW to theC-terminus of the MC1R specific ligand via lysine-mercaptopropionic acidlinker (named MC1RL-800). As determined by the europium time-resolvedfluorescence competition binding assay using Eu-NDP-α-MSH, the MC1RL-800probe displays high in vitro binding affinity to MC1R in A375/MC1R cells(Ki 0.4±0.1 nM), a slightly weaker avidity than unmodified peptide (0.17nM). The in vitro microscopic results showed binding of the probe on thecell membrane as early as 15 seconds after adding the probe.

To determine the uptake profile of the probe to the tumor cellsimmediately after injection, in vivo, intravital confocal imaging of adorsal skin-fold window chamber tumor model was used. The window chambermodel has been used for high-resolution imaging using wide-fieldfluorescence, transmission or reflectance imaging, as well asepifluorescence, confocal and multiphoton/nonlinear microscopy (41). Theinventors' results showed internalization of the probes at 24 h afterinjection, which makes the ligand a good candidate for radionuclidelabeling and radiotherapy.

MC1R expression ranges from several hundred to around 10,000 receptorsper cell in different human cell lines (21). Therefore, development of anon-invasive imaging method for visualizing and quantifying MC1Rexpression will be useful for monitoring alteration in MC1R expressionas an indicator of treatment response (56). The inventors' in vivoresults indicate that MC1RL-800 imaging probe is able to differentiatedifferent levels of MC1R expression in A375 melanoma tumors with lowlevels of MC1R (400±93 sites/cell, (38) and A375/MC1R tumors with highlevels of expression (75,000 sites/cell). In addition, co-injection ofNDP-a-MSH and MC1RL-800 probe significantly decreased the probe-relatedfluorescence signal (P<0.05) in both A375 and A375/MC1R tumor, showingthe specific recognition of MC1R by the probe in the tumors.

Here, the inventors used whole animal imaging and ex vivo imaging oftumors and organs to study MC1RL-800 probe biodistribution and both werein agreement and fit in the inventors' mathematical model. Therefore,near infrared fluorescent imaging provides high sensitivity fordetection, visualization, and quantification of fluorescence distributedthroughout the body of living mice and consequently, there is no need tosacrifice multiple animals at multiple time points for this type ofstudy.

The in vivo biodistribution of MC1RL-800 indicates high tumor and kidneyuptake as early as 15 min after injection. While the inventors showhigher accumulation of the probe in kidneys compared to MC1R highexpressing tumor at early time points, the probe was completely clearedfrom both kidneys and tumor at the same rate by 72 hr after injectionbased on the inventors' experimental as well as simulation data.Co-injection of MC4R/5R-selective ligand is shown to significantlydecrease the nonspecific accumulation of the probe in the kidneys.MC4R/5R-selective ligand has high affinity against MC5R which isexpressed in the kidneys (43). The results here confirmed highspecificity of this compound against MC5R. Thus, it appears that therenal uptake of MC1RL-800 is due to off-target binding and can bepharmacologically blocked. In addition, the inventors' study showed thatthe lower dose of 1 nmol/kg provides greater discrimination and fasterrenal clearance compared to the higher dose at 5 nmol/Kg,re-demonstrating its high affinity to MC1R compared to MC5R. Therefore,kidney accumulation of the probe can be reduced either by co-injectionof MC4R/5R-selective ligand or by using lower doses of the MC1RL-800probe. Although, kidney accumulation should not be a serious problem inuse of the probe for detection of regional lymph node metastases, sinceit will be administered peritumorally and clear through the lymphaticswhere macrophages will likely ingest the probe decreasing systemiccirculation. Also, since the probe clears within days afteradministration, toxicity to the kidneys should be minimal when deliveredsystemically for detection of distal metastases.

In summary, to the best of the inventors' knowledge, this represents thefirst account in which MC1R has been validated as a potential target inboth melanoma cell lines (through DNA microarray) and patient samples(through DNA microarray and IHC). The inventors' results have indicatedthat, while MC1R is highly expressed in 40%-67% of melanomas, previousreports suggesting high expression in 80% of melanomas may have been anover-estimate. In addition, the specific MC1R fluorescent imaging probewith high affinity to the MC1R was successfully synthesized andcharacterized. The uptake study of the probe was carried out in vitro aswell as in vivo. The inventors' study demonstrates that the probe candifferentiate high- from low-MC1R expressing tumors. Therefore,MC1RL-800 is a promising molecular probe for imaging of MC1R-positivemelanoma and MC1R expression. Despite the promising tumor-targetingproperties of the probe, its biodistribution profile could still beimproved. Lower kidney uptake could be obtained by decreasing affinityfor MC5R by modifying the physicochemical properties or peptidomimeticstructure of the ligand.

Since the boundaries of lesions often cannot be reliably determined inpatients with melanoma, repeated surgical excisions are frequentlyrequired to achieve tumor-free margins (57). Invisible NIR fluorescentlight with high resolution and sensitivity is used for real-timeintraoperative image-guidance during surgery (58). The targeted agentsof the invention (e.g., fluorescent targeted agents) can also offer anopportunity for image guided surgery for assessment of tumor margins inmelanoma patients.

In conclusion, the imaging probe developed in this study is useful fortranslation as a clinical PET tracer for noninvasive identification ofregional lymph node metastasis when injected locally at the primarylesion site as well as detection of distal metastases in malignantmelanomas. In addition, it could have particular benefit for theevaluation of therapeutic efficacy. In the future, this ligand couldalso be used in clinic as a MC1R targeted delivery vehicle forradionuclides, toxins, and chemotherapeutic molecules.

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Materials and Methods for Example 3

Online Methods

Synthesis of Gd-Tx.

Information on the synthesis of Gd-Tx, including mass spectral data, canbe found in the Supporting Information.

Crystalization of Gd-Tx and Determination of Structure.

Crystals suitable for X-ray diffraction were obtained by dissolvingGd-Tx (2 mg, 2.26 μmol) in 1 mL methanol. Sodium nitrate (0.2 mg, 4equiv.) was added and the solution was heated to reflux at 60° C. for 24hours. At this point, 0.25 mL chloroform was added and the solution wasplaced in a vial and diethyl ether was allowed to slowly diffuse intothe solution at 5° C. For full crystallographic data, please seeSupporting Information. Further details of the structure may also beobtained from the Cambridge Crystallographic Data Centre by quoting CCDCnumber 859294.

Synthesis of Targeted Triblock Polymers.

IVECT™ triblock polymers with a terminal azide were obtained fromIntezyne Technologies (Tampa, Fla.) and either NDP-α-MSH-lys-hexyne 2 or1⁴⁰ (Scheme 1) were used as the MC1R-selective ligand. Standard clickchemistry was conducted as previously published.⁴⁰

Formulation and Stabilization of Gd-Tx Micelles.

For targeted formulations, 5-10% of the targeted polymer and 90-95% ofthe untargeted polymer were used, respectively. Gd-Tx (0.05-5% w/w), wasdissolved in dimethylsulfoxide (DMSO, 380 μL). The triblock polymer (750mg) was dissolved in water (150 mL) at a concentration of 5 mg/mL andstirred with slight heating until fully dissolved. After cooling to roomtemperature, the polymer solution was placed in a sheer mixer and theGd-Tx solution was added. The resulting solution was then passed througha microfluidizer (Microfluidics M-110Y) at 23,000 PSI, filtered througha 0.22 μm Steriflip-GP Filter Unit (Millipore) and lyophilized.

For stabilized formulations, micelles were subject to anFe(III)-mediated crosslinking reaction.¹² FeCl₃ was prepared atconcentration of 1.35 g/mL in 20 mM Tris-Cl (pH 7.4). The targeted anduntargeted micelles were then dissolved in the Fe(III)-tris solution ata concentration of 20 mg/mL and the solution was adjusted to pH 8through the dropwise addition of 0.1-1 M aqueous NaOH. The crosslinkingreaction was stirred for 12 hours. The contents of the reaction vesselwere then lyophilized.

Cell Culture.

HCT116 cells overexpressing hMC1R were engineered in our lab. HCT116cells were transfected with the pCMV6-Entry Vector (Origene; RC 203218)using the Fugene 6 transfection reagent (Roche; 1814-443). Transfectedcells were grown in a selection media containing 0.4 mg/ml geneticin(Life Technologies; 11811-031) and tested for the hMC1R cell surfaceexpression by saturation ligand binding assay.²³ Cells were maintainedunder standard conditions (37° C. and 5% CO₂) and were grown inDulbecco's modified Eagle medium (DMEM) supplemented with 10% FBS and 5%penicillin/streptomycin. For HCT116/hMC1R cells, geneticin (G418S, 0.8%)was added to the media to ensure proper selection. hMC1R expression wasverified through immunohistochemistry (IHC, see SupplementalInformation).

Europium Binding Assays. Europium binding assays were conducted aspreviously published.^(33,40)

In Vivo Murine Tumor Models.

All animal experiments were approved by institution review boardguidelines on the care and use of animals in research.HCT116/hMC1R-expressing tumor models were studied in female SCID/beigemice obtained from Harlan Laboratories at 6-8 weeks of age. HCT116/hMC1Rcells were injected at concentrations of 3×10⁶-10×10⁶ cells per 0.1 mLof phosphate-buffered saline. Tumor volume measurements were madebi-weekly and calculated by multiplying the length by the width squaredand dividing by two. Final volume measurements were determined throughROI analysis on the MRI.

MRI Imaging and Analysis.

All imaging was completed on a 7 Tesla, 30 cm horizontal bore Agilentmagnetic resonance imaging spectrometer ASR310 (Agilent Life SciencesTechnologies, Santa Clara, Calif. Detailed information on the MRIimaging and analysis, including imaging processing, can be found in theSupporting Information. Briefly, SCOUT images were acquired for sliceselection for both in vitro and in vivo imaging. For in vitro phantom T1studies, multiple TR SEMS (spin echo) imaging was performed to calculateT1 values. For in vivo imaging and quantification of tumor enhancementdue to uptake of the micelles, T1 weighted spin echo multi slice (SEMS)images were acquired, and intensity histograms for right (R) and left(L) whole tumors, and R and L whole kidney and liver were prepared usingthe MATLAB program (Mathworks, Natick, Mass.) for each time point. Thiswas done by drawing an ROI across all applicable slices. The meanintensity value for each time point was normalized to the mean intensityof the thigh muscle. A percent change value was then calculated bycomparing each time point after injection to the normalizedpre-injection intensity mean. The % change values for all tumors in agiven group (n=3 for all groups) were averaged to obtain the “mean tumor% change” at time points from 1 to 48 h. Percent change values were alsoaveraged for R and L kidney to obtain the “mean kidney % change” values.

Supporting Information for Example 3

Synthesis of Gd-Tx 6.

All chemicals were obtained from commercial sources (Fisher Scientific,Acros Chemicals, Sigma-Aldrich or Strem Chemicals) and used as suppliedunless otherwise noted. All solvents were of reagent grade quality.Fisher silica gel (230-400 mesh, Grade 60 Å) and Sorbent Technologiesalumina (neutral, standard activity I, 50-200 μm) were used for columnchromatography. Thin layer chromatography (TLC) analyses were eitherperformed on silica gel (aluminum backed, 200 μm or glass backed, 250μm) or alumina neutral TLC plates (polyester backed, 200 μm), bothobtained from Sorbent Technologies. Low- and high-resolution ESI massspectra (MS) were obtained at the Mass Spectrometry Facility of theDepartment of Chemistry and Biochemistry at The University of Texas atAustin using a Thermo Finnigan LTQ instrument and a Qq FTICR (7 Tesla)instrument, respectively. HPLC spectra were taken on a Shimadzu HighPerformance Liquid Chromatograph (Fraction Collector Module FRC-10A,Auto Sampler SIL-20A, System Controller CBM-20A, UV/Vis Photodiode ArrayDetector SPD-M20A, Prominence). The tripyrrane dialdehyde species 5(generally referred to as “TP-4”) was provided by Pharmacyclics Inc. andsynthesized as previously described.¹ The precursor1,2-dimethoxy-4,5-dinitrobenzene 4 was synthesized as previouslydescribed.²

The gadolinium complex used in this study (Gd-Tx 6) was prepared asshown in Scheme 51. Briefly, compound 4 (1 g, 4.38 mmol) was dissolvedin 10 mL methanol and placed in a hydrogenation flask. The solution waspurged with nitrogen for 5 minutes and palladium on activated carbon(10%, 0.1 g) was added. The mixture was degassed and allowed to reactwith hydrogen gas at 100 psi with agitation for 18 hours, filtered underSchlenk conditions through a minimal pad of Celite, and added instantlyto a solution of TP-4 5 (2.11 g, 4.38 mmol) in 15 mL methanol undernitrogen at 70° C. At this point, aqueous hydrochloric acid was added (2mL, 0.5 M) and the deep red reaction mixture was stirred for 4 hours.Next, gadolinium acetate tetrahydrate (2.67 g, 6.57 mmol, 1.5 equiv) wasadded together with 3 ml triethylamine and the solution was stirred at70° C. for 16 hours, during which time the solution gradually changedcolor from deep red to deep green. The solvent was removed in vacuo andthe residue was then subjected to column chromatography (silica gel). Toremove apolar impurities, the column was eluted first with a mixture of95% CH₂Cl₂ and 5% MeOH. The product slowly starts to elute when amixture of 60% CH₂Cl₂ and 40% MeOH is used as the eluent. The deep greenfraction isolated using this eluent mixture was collected and thesolvent was removed in vacuo to give 6 (Gd-Tx) as a deep greencrystalline material (1.63 g, 42%). UV/Vis (MeOH, 25° C.): λ_(max)=470(Soret-type band); 739 (Q-type band); Low Resolution MS (ESI in MeOH):797.25 (M⁺-2OAc+OMe), 825.42 (M⁺-OAc). High Resolution MS (ESI in MeOH):calculated for C₃₈H₄₅N₅O₆Gd⁺¹=825.2611; found: 825.2621(C₃₈H₄₅N₅O₆Gd⁺¹,M⁺-OAc).

MRI Imaging and Analysis.

All imaging was completed on a 7 Tesla, 30 cm horizontal bore Agilentmagnetic resonance imaging spectrometer ASR310 (Agilent Life SciencesTechnologies, Santa Clara, Calif.). In vitro imaging of Gd-Tx micellephantoms was completed using a SCOUT image for slice selection, and amultiple TR SEMS (spin echo) image was performed in order to calculatethe T₁ values. The TR calculation sequence consisted of TR values of 20,10.99, 6.03, 3.31, 1.82, 1.00, 0.55, 0.30, 0.17, 0.09 and 0.05 s; the TEwas 8.62 ms, the data matrix was 128×128, 4 averages, 2 dummy scans, FOVwas 80 mm×40 mm or 40 mm×90 mm and the slice thickness was 1-2 mm(depending on the phantom). The T₁ values were calculated using theVnmrJ software (Agilent Life Sciences Technologies, Santa Clara,Calif.), and values were verified using MATLAB (Mathworks, Natick,Mass.).

Once the tumors in the animals reached an average of ˜500 mm³, theanimals were pair-matched by tumor size and sorted into four groupswhich would receive the following micelles: TG,XL; UT-XL; T-UXL; orUT-UXL. Each animal was imaged the day before micelle injection for“pre” images. The following morning, each animal was individuallyadministered 12 μmol/kg Gd-Tx (as Gd-Tx micelles) dissolved in 200 uLsaline, via tail vein injection, and the time of injection was noted.Follow-up MRI images were taken at 1 hr, 4 hr, 12 hr, 24 and 48 h afterinjection of the micelles.

All animals were sedated using isoflurane and remained under anesthesiafor the duration of the imaging. Animals were kept at body temperature(˜37° C.) using a warm air blower; the temperature of the air wasadjusted to maintain the body temperature and was monitored using afiber optic rectal probe. SCOUT images were taken to determine animalposition within the magnet and setup the slices for the T₁ weighted spinecho multi slice (SEMS) images. The SEMS images were taken as coronal-90images (read direction along the X-axis, phase-encode along the Z-axis),with data matrix of 128×128 and a FOV of 40 mm (read)×90 mm (phase); 15one-mm thick slices were taken with a 0.5 mm gap between slices; the TRwas 180 ms, and TE was 8.62 ms; there were 8 averages taken for eachimage, resulting in a total scan time of about 3 minutes per SEMS image.

Images were processed using MATLAB (Mathworks, Natick, Mass.) to drawregions of interest (ROI) in the tumors, kidney, liver and thigh muscleover multiple slices for each mouse at each time point. All intensitiesfor each area of interest were averaged to determine a mean intensity.The mean intensity of each area was then normalized to the meanintensity of the thigh to generate a normalized intensity (NI):

${NI} = \frac{I_{tumor}}{I_{thigh}}$A percent change value was then calculated by comparing each normalizedtime point after injection to the normalized pre-injection intensitymean:

${\%\mspace{20mu}{Change}} = {\frac{{NI}_{12h}}{{NI}_{pre}} \times 100}$

Since the right and left tumors are histologically equivalent (FigureS.4), the % change values for all tumors were averaged to obtain an“average tumor % change” at time points 1-24 h. Percent change valueswere also averaged for R and L kidney to obtain an “average kidney %change” at time points 1-24 h.

X-Ray Experimental for (C₃₆H₄₂N₅O₄)Gd(NO₃)₂—CH₃OH—H₂O 6.

Crystals grew as dark green prisms by slow diffusion of diethyl etherinto a solution of 6 dissolved in methanol/chloroform (4:1) and sodiumnitrate (4 equiv.). The data crystal had approximate dimensions:0.23×0.07×0.07 mm. The data were collected on a Nonius Kappa CCDdiffractometer using a graphite monochromator with MoKa radiation(λ=0.71073 Å). A total of 384 frames of data were collected usingw-scans with a scan range of 1.2° and a counting time of 144 seconds perframe. The data were collected at 153 K using an Oxford Cryostream lowtemperature device. Details of crystal data, data collection andstructure refinement are listed in Table 7. Data reduction wereperformed using DENZO-SMN. The structure was solved by direct methodsusing SIR97 ³ and refined by full-matrix least-squares on F² withanisotropic displacement parameters for the non-H atoms usingSHELXL-97.⁴ The hydrogen atoms were calculated in ideal positions withisotropic displacement parameters set to 1.2× Ueq of the attached atom(1.5× Ueq for methyl hydrogen atoms).

The function, Σw(|F_(O)|²−|F_(C)|²)², was minimized, wherew=1/[(s(F_(O)))²+(0.0313*P)²+(3.8824*P)] and P=(|F_(O)|²+2|F_(C)|²)/3.R_(W)(F²) refined to 0.103, with R(F) equal to 0.0537 and a goodness offit S=1.17. Definitions used for calculating R(F),R_(W)(F²) and thegoodness of fit, S, are given below. * The data were checked forsecondary extinction but no correction was necessary. Neutral atomscattering factors and values used to calculate the linear absorptioncoefficient are from the International Tables for X-ray Crystallography(1992).⁵ All figures were generated using SHELXTL/PC.6 Tables ofpositional and thermal parameters, bond lengths and angles, torsionangles and figures are included in the tables below.

* R_(W)(F²)={Sw(|F_(O)|²−|F|_(C) ²)²/Sw(|F₀|)⁴}^(1/2) where w is theweight given each reflection. R(F)=S(|F_(O)|−|F_(C)|)/S|F_(O)|} forreflections with F_(O)>4(s(F_(O))).S=[Sw(|F_(O)|²−|C_(C)|²)²/(n−p)]^(1/2), where n is the number ofreflections and p is the number of refined parameters.

TABLE 7 Crystal data and structure refinement for 6. Empirical formulaC37 H48 Gd N7 O12 Formula weight 940.07 Temperature 123(2) K. Wavelength0.71069 Å Crystal system Monoclinic Space group P21/c Unit celldimensions a = 15.3250(10) Å a = 90°. b = 11.5950(8) Å b = 99.592(2)°. c= 21.5387(15) Å g = 90°. Volume 3773.8(4) Å³ Z 4 Density (calculated)1.655 Mg/m³ Absorption coefficient 1.832 mm⁻¹ F(000) 1916 Crystal size0.23 × 0.07 × 0.07 mm Theta range for data collection 2.00 to 25.00°.Index ranges −18 <= h <= 18, −13 <= k <= 12, −25 <= l <= 25 Reflectionscollected 11918 Independent reflections 6623 [R(int) = 0.0816]Completeness to theta = 25.00° 99.9 % Absorption correctionSemi-empirical from equivalents Max. and min. transmission 1.00 and0.869 Refinement method Full-matrix least-squares on F²Data/restraints/parameters 6623/1/523 Goodness-of-fit on F² 1.171 FinalR indices [I > 2sigma(I)] R1 = 0.0537, wR2 = 0.0838 R indices (all data)R1 = 0.1176, wR2 = 0.1031 Largest diff. peak and hole 1.759 and −0.791e.Å⁻³ * R_(w)(F²) = {Sw(|F_(O)|²-|F|_(C) ²)²/Sw(|F_(O))⁴}^(1/2) where wis the weight given each reflection. R(F) = S(|F_(O)|-|F_(C)|)/S|F_(O)|}for reflections with F_(O) > 4(s(F_(O))). S =[Sw(|F_(O)|²-|F_(C)|²)²/(n-p)]^(1/2), where n is the number ofreflections and p is the number of refined parameters.

TABLE 8 Atomic coordinates (×10⁴) and equivalent isotropic displacementparameters (Å² × 10³) for 6. U(eq) is defined as one third of the traceof the orthogonalized U^(ij) tensor. x y z U(eq) Gd1   2938(1)   2064(1)5736(1) 23(1) N1   2083(3)   3925(4) 5489(2) 17(1) N2   3053(3)  2448(5) 4665(2) 24(1) N3   3286(3)    289(5) 5126(2) 22(1) N4  2637(3)    119(4) 6196(2) 19(1) N5   1931(3)   2166(5) 6471(2) 22(1)O1   4487(3)   6406(4) 3226(2) 45(1) O2   4064(3) −3804(4) 5297(2) 29(1)O3   3469(3) −3945(4) 6334(2) 26(1) O4   1365(3)   5187(4) 9116(2) 36(1)O5   3875(3)   3725(4) 6020(2) 40(1) C1   1631(4)   4533(6) 5874(3)23(2) C2   1404(4)   5691(5) 5629(3) 22(2) C3   1739(4)   5767(5)5087(3) 21(2) C4   2168(4)   4675(6) 5001(3) 22(2) C5   2587(4)  4440(5) 4486(3) 20(2) C6   2986(4)   3440(6) 4317(3) 23(2) C7  3341(4)   3225(6) 3735(3) 25(2) C8   3587(4)   2099(6) 3745(3) 26(2)C9   3414(4)   1636(6) 4331(3) 26(2) C10   3525(4)    491(6) 4579(3)27(2) C11   3361(4)  −784(6) 5424(3) 21(2) C12   3739(4) −1768(6)5191(3) 27(2) C13   3748(4) −2802(6) 5493(3) 24(2) C14   3413(4)−2886(6) 6075(3) 21(1) C15   3053(4) −1936(6) 6318(3) 21(2) C16  3009(4)  −874(6) 5994(3) 22(2) C17   2212(4)    152(5) 6675(3) 22(2)C18   1851(4)   1219(6) 6826(3) 23(2) C19   1332(4)   1490(6) 7317(3)22(2) C20   1135(4)   2633(6) 7253(3) 22(2) C21   1492(4)   3037(6)6707(3) 23(2) C22   1381(4)   4127(5) 6433(3) 24(2) C23    886(4)  6586(6) 5915(3) 26(2) C24  −107(4)   6433(6) 5729(3) 34(2) C25  1670(4)   6785(5) 4640(3) 24(2) C26    921(4)   6688(5) 4084(3) 28(2)C27   3445(4)   4109(6) 3239(3) 24(2) C28   4400(5)   4337(6) 3187(3)33(2) C29   4518(5)   5430(6) 2832(3) 36(2) C30   3970(6)   1440(6)3255(3) 44(2) C31   4383(5) −3745(6) 4708(3) 38(2) C32   3192(4)−4069(6) 6939(3) 27(2) C33   1115(4)    687(6) 7810(3) 31(2) C34   643(5)   3360(6) 7661(3) 32(2) C35   1266(4)   3917(6) 8210(3) 28(2)C36    790(5)   4700(6) 8600(3) 30(2) O1A   4559(3)   1526(4) 5890(2)43(1) O2A   3990(4)   1671(5) 6738(2) 47(2) O3A   5357(4)   1090(5)6795(3) 69(2) N1A   4653(5)   1426(5) 6479(4) 45(2) O1B   1570(4)−1828(5) 2746(3) 58(2) O2B   2559(4) −1697(5) 3599(2) 54(2) O3B  2650(4) −3059(5) 2915(2) 54(2) N1B   2254(5) −2175(6) 3088(3) 46(2)O1C   1533(3)   1345(4) 5107(2) 30(1) C1C    696(5)   1179(7) 5306(3)50(2)

TABLE 9 Bond lengths [Å] and angles [°] for 6. Gd1-N2 2.384(5) O5-H1WA0.81 Gd1-N5 2.391(5) O5-H1WB 0.81 Gd1-O5 2.419(5) C1-C22 1.406(9)Gd1-O1C 2.488(4) C1-C2 1.463(9) Gd1-O2A 2.511(5) C2-C3 1.356(8) Gd1-O1A2.529(5) C2-C23 1.498(8) Gd1-N1 2.534(5) C3-C4 1.452(9) Gd1-N4 2.536(5)C3-C25 1.515(8) Gd1-N3 2.545(5) C4-C5 1.397(9) Gd1-N1A 2.935(7) C5-C61.387(9) N1-C1 1.362(8) C5-H5 0.95 N1-C4 1.388(8) C6-C7 1.471(9) N2-C91.359(8) C7-C8 1.358(9) N2-C6 1.367(8) C7-C27 1.507(8) N3-C10 1.312(8)C8-C9 1.435(8) N3-C11 1.395(8) C8-C30 1.499(9) N4-C17 1.310(7) C9-C101.431(9) N4-C16 1.386(8) C10-H10 0.95 N5-C18 1.356(8) C11-C12 1.410(8)N5-C21 1.358(8) C11-C16 1.424(8) O1-C29 1.419(8) C12-C13 1.363(9) O1-H10.8400 C12-H12 0.95 O2-C13 1.352(8) C13-C14 1.435(8) O2-C31 1.435(7)C14-C15 1.374(9) O3-C14 1.346(7) C15-C16 1.412(9) O3-C32 1.443(7)C15-H15 0.95 O4-C36 1.415(7) C17-C18 1.415(9) O4-H4 0.84 C17-H17 0.95C18-C19 1.459(8) C30-H30C 0.98 C19-C20 1.361(8) C31-H31A 0.98 C19-C331.491(9) C31-H31B 0.98 C20-C21 1.454(8) C31-H31C 0.98 C20-C34 1.508(9)C32-H32A 0.98 C21-C22 1.393(9) C32-H32B 0.98 C22-H22 0.95 C32-H32C 0.98C23-C24 1.519(9) C33-H33A 0.98 C23-H23A 0.99 C33-H33B 0.98 C23-H236 0.99C33-H33C 0.98 C24-H24A 0.98 C34-C35 1.535(9) C24-H24B 0.98 C34-H34A 0.99C24-H24C 0.98 C34-H34B 0.99 C25-C26 1.519(8) C35-C36 1.505(9) C25-H25A0.99 C35-H35A 0.99 C25-H25B 0.99 C35-H35B 0.99 C26-H26A 0.98 C36-H36A0.99 C26-H26B 0.98 C36-H36B 0.99 C26-H26C 0.98 O1A-N1A 1.257(8) C27-C281.510(9) O2A-N1A 1.271(8) C27-H27A 0.99 O3A-N1A 1.239(7) C27-H27B 0.99O1B-N1B 1.243(8) C28-C29 1.506(9) O2B-N1B 1.250(8) C28-H28A 0.99 O3B-N1B1.279(8) C28-H28B 0.99 O1C-C1C 1.430(8) C29-H29A 0.99 O1C-H1OC 0.80C29-H29B 0.99 C1C-H1C1 0.98 C30-H30A 0.98 C1C-H1C2 0.98 C30-H30B 0.98C1C-H1C3 0.98 N2-Gd1-N5 142.30(17) N2-Gd1-O5 87.69(17) N5-Gd1-O5102.37(17) O1A-Gd1-N3 65.33(16) N2-Gd1-O1C 74.53(16) N1-Gd1-N3136.53(15) N5-Gd1-O1C 77.36(15) N4-Gd1-N3 63.23(16) O5-Gd1-O1C145.86(15) N2-Gd1-N1A 111.7(2) N2-Gd1-O2A 136.46(18) N5-Gd1-N1A 105.6(2)N5-Gd1-O2A 80.37(17) O5-Gd1-N1A 67.83(16) O5-Gd1-O2A 69.99(16)O1C-Gd1-N1A 145.83(16) O1C-Gd1-O2A 141.28(16) O2A-Gd1-N1A 25.49(17)N2-Gd1-O1A 86.79(17) O1A-Gd1-N1A 25.21(17) N5-Gd1-O1A 130.79(17)N1-Gd1-N1A 135.54(16) O5-Gd1-O1A 68.56(16) N4-Gd1-N1A 76.57(17)O1C-Gd1-O1A 136.97(15) N3-Gd1-N1A 80.31(18) O2A-Gd1-O1A 50.67(17)Cl-N1-C4 104.9(5) N2-Gd1-N1 75.92(16) Cl-N1-Gd1 127.6(4) N5-Gd1-N174.26(16) C4-N1-Gd1 125.6(4) O5-Gd1-N1 68.89(15) C9-N2-C6 107.0(5)O1C-Gd1-N1 78.50(15) C9-N2-Gd1 119.1(4) O2A-Gd1-N1 124.78(17) C6-N2-Gd1132.6(4) O1A-Gd1-N1 134.41(16) C10-N3-C11 124.0(6) N2-Gd1-N4 126.27(16)C10-N3-Gd1 115.6(4) N5-Gd1-N4 66.79(17) C11-N3-Gd1 119.5(4) O5-Gd1-N4138.40(15) C17-N4-C16 123.8(5) O1C-Gd1-N4 73.58(15) C17-N4-Gd1 115.4(4)O2A-Gd1-N4 68.62(16) C16-N4-Gd1 120.3(4) O1A-Gd1-N4 88.16(17) C18-N5-C21107.1(5) N1-Gd1-N4 135.87(16) C18-N5-Gd1 117.6(4) N2-Gd1-N3 66.22(17)C21-N5-Gd1 134.4(4) N5-Gd1-N3 126.54(17) C29-O1-H1 109.5 O5-Gd1-N3127.43(16) C13-O2-C31 115.3(5) O1C-Gd1-N3 71.67(15) C14-O3-C32 117.2(5)O2A-Gd1-N3 97.85(18) C36-O4-H4 109.5 Gd1-O5-H1WA 110.8 N3-C10-H10 121.0Gd1-O5-H1WB 130.8 C9-C10-H10 121.0 H1WA-O5-H1WB 115.5 N3-C11-C12124.4(6) N1-C1-C22 125.9(6) N3-C11-C16 116.6(6) N1-C1-C2 111.8(5)C12-C11-C16 119.0(6) C22-C1-C2 122.3(6) C13-C12-C11 120.9(6) C3-C2-C1105.5(5) C13-C12-H12 119.5 C3-C2-C23 127.7(6) C11-C12-H12 119.5C1-C2-C23 126.7(6) O2-C13-C12 125.6(6) C2-C3-C4 107.3(5) O2-C13-C14114.5(6) C2-C3-C25 127.0(6) C12-C13-C14 119.9(6) C4-C3-C25 125.7(6)O3-C14-C15 125.1(5) N1-C4-C5 126.3(6) O3-C14-C13 114.6(6) N1-C4-C3110.4(5) C15-C14-C13 120.3(6) C5-C4-C3 123.3(6) C14-C15-C16 120.0(6)C6-C5-C4 130.7(6) C14-C15-H15 120.0 C6-C5-H5 114.6 C16-C15-H15 120.0C4-C5-H5 114.6 N4-C16-C15 123.9(5) N2-C6-C5 123.7(6) N4-C16-C11 116.3(6)N2-C6-C7 108.7(6) C15-C16-C11 119.7(6) C5-C6-C7 127.4(6) N4-C17-C18118.1(6) C8-C7-C6 106.9(6) N4-C17-H17 120.9 C8-C7-C27 127.2(6)C18-C17-H17 120.9 C6-C7-C27 125.9(6) N5-C18-C17 120.2(6) C7-C8-C9106.4(6) N5-C18-C19 110.3(6) C7-C8-C30 128.2(6) C17-C18-C19 129.5(6)C9-C8-C30 125.4(6) C20-C19-C18 105.9(6) N2-C9-C10 118.5(6) C20-C19-C33127.5(6) N2-C9-C8 111.0(6) C18-C19-C33 126.5(6) C10-C9-C8 130.4(6)C19-C20-C21 106.8(6) N3-C10-C9 118.0(6) C19-C20-C34 127.5(6) C21-C20-C34125.7(6) C7-C27-C28 112.9(5) N5-C21-C22 123.3(6) C7-C27-H27A 109.0N5-C21-C20 109.9(6) C28-C27-H27A 109.0 C22-C21-C20 126.8(6) C7-C27-H27B109.0 C21-C22-C1 129.5(6) C28-C27-H27B 109.0 C21-C22-H22 115.3H27A-C27-H27B 107.8 C1-C22-H22 115.3 C29-C28-C27 112.6(6) C2-C23-C24112.8(5) C29-C28-H28A 109.1 C2-C23-H23A 109.0 C27-C28-H28A 109.1C24-C23-H23A 109.0 C29-C28-H28B 109.1 C2-C23-H23B 109.0 C27-C28-H28B109.1 C24-C23-H23B 109.0 H28A-C28-H28B 107.8 H23A-C23-H23B 107.8O1-C29-C28 110.5(6) C23-C24-H24A 109.5 O1-C29-H29A 109.6 C23-C24-H24B109.5 C28-C29-H29A 109.6 H24A-C24-H24B 109.5 O1-C29-H29B 109.6C23-C24-H24C 109.5 C28-C29-H29B 109.6 H24A-C24-H24C 109.5 H29A-C29-H29B108.1 H24B-C24-H24C 109.5 C8-C30-H30A 109.5 C3-C25-C26 114.0(5)C8-C30-H30B 109.5 C3-C25-H25A 108.8 H30A-C30-H30B 109.5 C26-C25-H25A108.8 C8-C30-H30C 109.5 C3-C25-H25B 108.8 H30A-C30-H30C 109.5C26-C25-H25B 108.8 H30B-C30-H30C 109.5 H25A-C25-H25B 107.6 O2-C31-H31A109.5 C25-C26-H26A 109.5 O2-C31-H31B 109.5 C25-C26-H26B 109.5H31A-C31-H31B 109.5 H26A-C26-H26B 109.5 O2-C31-H31C 109.5 C25-C26-H26C109.5 H31A-C31-H31C 109.5 H26A-C26-H26C 109.5 H31B-C31-H31C 109.5H26B-C26-H26C 109.5 O3-C32-H32A 109.5 O3-C32-H32B 109.5 C35-C36-H36A109.1 H32A-C32-H32B 109.5 O4-C36-H36B 109.1 O3-C32-H32C 109.5C35-C36-H36B 109.1 H32A-C32-H32C 109.5 H36A-C36-H36B 107.8 H32B-C32-H32C109.5 N1A-01A-Gd1 95.8(4) C19-C33-H33A 109.5 N1A-O2A-Gd1 96.3(4)C19-C33-H33B 109.5 O3A-N1A-O1A 121.8(8) H33A-C33-H33B 109.5 O3A-N1A-O2A121.0(7) C19-C33-H33C 109.5 O1A-N1A-O2A 117.2(6) H33A-C33-H33C 109.5O3A-N1A-Gd1 176.1(5) H33B-C33-H33C 109.5 O1A-N1A-Gd1 59.0(4) C20-C34-C35112.1(6) O2A-N1A-Gd1 58.3(3) C20-C34-H34A 109.2 O1B-N1B-O2B 122.2(7)C35-C34-H34A 109.2 O1B-N1B-O3B 118.7(7) C20-C34-H34B 109.2 O2B-N1B-O3B119.1(7) C35-C34-H34B 109.2 C1C-O1C-Gd1 128.3(4) H34A-C34-H34B 107.9C1C-O1C-H1OC 103.3 C36-C35-C34 112.8(6) Gd1-O1C-H1OC 115.7 C36-C35-H35A109.0 O1C-C1C-H1C1 109.5 C34-C35-H35A 109.0 O1C-C1C-H1C2 109.5C36-C35-H35B 109.0 H1C1-C1C-H1C2 109.5 C34-C35-H35B 109.0 O1C-C1C-H1C3109.5 H35A-C35-H35B 107.8 H1C1-C1C-H1C3 109.5 O4-C36-C35 112.5(6)H1C2-C1C-H1C3 109.5 O4-C36-H36A 109.1

TABLE 10 Anisotropic displacement parameters (Å² × 10³) for 6. Theanisotropic displacement factor exponent takes the form: −2p²[h²a*²U¹¹ +. . . + 2 h k a* b* U¹²]. U¹¹ U²² U³³ U²³ U¹³ U¹² Gd1 29(1) 19(1) 22(1)   0(1)    8(1)    1(1) N1 13(3) 19(3) 19(3)  −2(3)    0(2)    0(2) N220(3) 22(3) 28(3)    0(3)    6(3)    2(2) N3 31(3) 21(3) 16(3)    3(3)   8(3)    3(3) N4 31(3) 16(3) 13(3)  −2(2)    8(2)    1(3) N5 33(3)11(3) 20(3)    1(3)    2(2)    1(3) O1 41(3) 31(3) 61(3)  −7(3)    1(3) −2(3) O2 45(3) 22(3) 25(2)    1(2)   18(2)    8(2) O3 34(3) 16(3) 28(3)   3(2)    9(2)    6(2) O4 45(3) 35(3) 28(3)  −2(2)    9(2)  −5(3) O527(3) 24(3) 63(3)    1(3)  −4(2)    6(2) C1 27(4) 23(4) 18(4)  −5(3)   1(3)  −5(3) C2 24(4) 16(4) 25(4)  −7(3)    0(3)    3(3) C3 18(4)21(4) 22(4)    1(3)    2(3)    2(3) C4 25(4) 19(4) 21(4)    0(3)  −1(3) −2(3) C5 30(4) 16(4) 14(3)    6(3)  −2(3)  −6(3) C6 23(4) 23(4) 22(4) −1(3)    4(3)  −3(3) C7 22(4) 28(5) 24(4)  −1(3)    3(3)  −2(3) C835(4) 26(4) 20(3)    2(3)   14(3)  −5(4) C9 35(4) 21(4) 23(4)    0(3)   8(3)  −2(3) C10 32(4) 24(4) 29(4)  −5(3)   14(3)    4(3) C11 27(4)19(4) 18(3)  −1(3)    5(3)  −3(3) C12 25(4) 35(5) 22(4)  −2(3)    8(3)   2(3) C13 25(4) 25(4) 21(4)  −4(3)    2(3)    2(3) C14 26(4) 15(4)23(3)    2(3)    3(3)  −3(3) C15 28(4) 22(4) 15(3)    2(3)    6(3)   3(3) C16 27(4) 21(4) 16(3)  −1(3)    2(3)    0(3) C17 28(4) 13(4)23(4)    2(3)  −1(3)  −5(3) C18 28(4) 22(4) 18(3)    0(3)    6(3)   4(3) C19 22(4) 25(4) 21(4)  −1(3)    7(3)  −1(3) C20 31(4) 30(5) 7(3)  −5(3)    7(3)    3(3) C21 30(4) 26(4) 14(3)  −5(3)    5(3)  −1(4)C22 33(4) 16(4) 23(4)  −5(3)    7(3)    0(3) C23 29(4) 22(4) 29(4)   5(3)   11(3)    6(3) C24 34(5) 30(5) 41(4)    3(4)   13(4)    4(4)C25 27(4) 23(4) 24(4)    1(3)    8(3)    5(3) C26 30(4) 22(4) 31(4)   6(3)    1(3)    1(3) C27 31(4) 22(4) 21(4)    4(3)    8(3)    3(3)C28 36(5) 35(5) 28(4)    2(3)   13(3)    2(4) C29 39(5) 32(5) 41(5) −3(4)   15(4)  −6(4) C30 86(7) 29(5) 25(4)    1(4)   26(4)   10(4) C3149(5) 27(5) 41(4)  −5(4)   22(4)   12(4) C32 40(4) 21(4) 21(4)    3(3)   5(3)    2(3) C33 30(4) 39(5) 26(4)    1(3)   16(3)    6(4) C34 47(5)23(4) 31(4)    7(3)   18(4)  −3(4) C35 34(4) 21(4) 29(4)  −5(3)    8(3)   4(3) C36 42(5) 19(4) 29(4)  −2(3)    7(4)    6(4) O1A 42(3) 43(4)44(3) −12(3)    5(3)    4(3) O2A 41(3) 62(4) 38(3)   16(3)    2(3)−11(3) O3A 50(4) 44(4) 100(5)    12(4) −25(4)   10(3) N1A 46(5) 19(4)65(5)    9(4)  −7(4)  −2(3) O1B 54(4) 62(5) 58(4)    8(3)    8(3)   8(3) O2B 70(4) 56(4) 41(3) −12(3)   22(3)  −4(3) O3B 65(4) 38(4)63(4)  −5(3)   26(3)   10(3) N1B 56(5) 43(5) 47(4)    3(4)   33(4) −5(4) O1C 32(3) 28(3) 31(3)  −4(2)    6(2)    3(2) C1C 45(5) 64(6)39(5) −12(4)  −3(4)    2(5)

TABLE 11 Hydrogen coordinates (×10⁴) and isotropic displacementparameters (Å² × 10³) for 6. x y z U(eq) H1 3987 6718 3140 68 H4 18005490 8988 54 H1WA 4334 3536 6246 59 H1WB 3757 4408 6021 59 H5 2602 50694206 24 H10 3764 −104 4355 33 H12 3992 −1710 4818 32 H15 2833 −1995 670426 H17 2149 −521 6915 26 H22 1095 4676 6658 28 H23A 1054 7360 5780 31H236 1044 6547 6379 31 H24A −269 6476 5270 51 H246 −413 7045 5923 51H24C −281 5680 5876 51 H25A 2236 6863 4479 29 H256 1585 7496 4878 29H26A 1047 6059 3808 42 H266 867 7414 3848 42 H26C 366 6530 4237 42 H27A3167 4841 3341 29 H276 3128 3839 2827 29 H28A 4753 4390 3615 39 H28B4630 3679 2970 39 H29A 5094 5410 2682 44 H29B 4044 5489 2460 44 H30A3858 1860 2855 67 H30B 3692 677 3200 67 H30C 4609 1351 3390 67 H31A 3929−3395 4389 56 H31B 4515 −4525 4574 56 H31C 4922 −3275 4758 56 H32A 3558−3575 7248 41 H32B 3259 −4875 7076 41 H32C 2570 −3841 6904 41 H33A 1497854 8212 46 H33B 1210 −110 7686 46 H33C 494 789 7857 46 H34A 207 28717830 39 H34B 313 3972 7400 39 H35A 1570 3302 8484 33 H35B 1724 4366 804133 H36A 501 5328 8330 36 H36B 322 4257 8759 36 H1OC 1576 791 4894 46H1C1 729 506 5585 76 H1C2 239 1051 4937 76 H1C3 548 1865 5533 76

TABLE 12 Torsion angles [°] for 6. N2-Gd1-N1-C1 177.5(5) N4-Gd1-N2-C9−34.6(5) N5-Gd1-N1-C1 20.8(5) N3-Gd1-N2-C9 −13.7(4) O5-Gd1-N1-C1−89.5(5) N1A-Gd1-N2-C9 54.4(5) O1C-Gd1-N1-C1 100.8(5) N5-Gd1-N2-C661.9(7) O2A-Gd1-N1-C1 −45.1(5) O5-Gd1-N2-C6 −45.4(6) O1A-Gd1-N1-C1−111.6(5) O1C-Gd1-N2-C6 105.1(6) N4-Gd1-N1-C1 49.4(5) O2A-Gd1-N2-C6−102.8(6) N3-Gd1-N1-C1 147.8(4) O1A-Gd1-N2-C6 −114.1(6) N1A-Gd1-N1-C1−75.7(6) N1-Gd1-N2-C6 23.4(5) N2-Gd1-N1-C4 −20.2(4) N4-Gd1-N2-C6160.6(5) N5-Gd1-N1-C4 −176.9(5) N3-Gd1-N2-C6 −178.5(6) O5-Gd1-N1-C472.8(5) N1A-Gd1-N2-C6 −110.4(6) O1C-Gd1-N1-C4 −96.9(5) N2-Gd1-N3-C1012.8(4) O2A-Gd1-N1-C4 117.2(4) N5-Gd1-N3-C10 151.4(4) O1A-Gd1-N1-C450.7(5) O5-Gd1-N3-C10 −54.1(5) N4-Gd1-N1-C4 −148.3(4) O1C-Gd1-N3-C1093.5(5) N3-Gd1-N1-C4 −49.9(5) O2A-Gd1-N3-C10 −124.8(5) N1A-Gd1-N1-C486.6(5) O1A-Gd1-N3-C10 −85.0(5) N5-Gd1-N2-C9 −133.3(4) N1-Gd1-N3-C1044.5(5) O5-Gd1-N2-C9 119.4(5) N4-Gd1-N3-C10 174.0(5) O1C-Gd1-N2-C9−90.1(5) N1A-Gd1-N3-C10 −106.3(5) O2A-Gd1-N2-C9 62.0(5) N2-Gd1-N3-C11−177.9(5) O1A-Gd1-N2-C9 50.7(5) N5-Gd1-N3-C11 −39.4(5) N1-Gd1-N2-C9−171.8(5) O5-Gd1-N3-C11 115.2(4) O1C-Gd1-N3-C11 −97.2(4) O1C-Gd1-N5-C1889.1(4) O2A-Gd1-N3-C11 44.4(5) O2A-Gd1-N5-C18 −59.0(4) O1A-Gd1-N3-C1184.3(4) O1A-Gd1-N5-C18 −53.7(5) N1-Gd1-N3-C11 −146.3(4) N1-Gd1-N5-C18170.5(5) N4-Gd1-N3-C11 −16.8(4) N4-Gd1-N5-C18 11.7(4) N1A-Gd1-N3-C1163.0(4) N3-Gd1-N5-C18 33.6(5) N2-Gd1-N4-C17 −149.9(4) N1A-Gd1-N5-C18−55.8(4) N5-Gd1-N4-C17 −11.1(4) N2-Gd1-N5-C21 −61.4(6) O5-Gd1-N4-C1771.4(5) O5-Gd1-N5-C21 41.1(5) O1C-Gd1-N4-C17 −94.1(4) O1C-Gd1-N5-C21−103.9(5) O2A-Gd1-N4-C17 77.3(4) O2A-Gd1-N5-C21 108.0(5) O1A-Gd1-N4-C17125.4(4) O1A-Gd1-N5-C21 113.3(5) N1-Gd1-N4-C17 −41.1(5) N1-Gd1-N5-C21−22.5(5) N3-Gd1-N4-C17 −171.4(5) N4-Gd1-N5-C21 178.7(6) N1A-Gd1-N4-C17102.8(4) N3-Gd1-N5-C21 −159.3(5) N2-Gd1-N4-C16 38.2(5) N1A-Gd1-N5-C21111.2(5) N5-Gd1-N4-C16 177.1(5) C4-N1-C1-C22 179.6(6) O5-Gd1-N4-C16−100.5(4) Gd1-N1-C1-C22 −15.2(9) O1C-Gd1-N4-C16 94.1(4) C4-N1-C1-C21.0(7) O2A-Gd1-N4-C16 -94.5(4) Gd1-N1-C1-C2 166.2(4) O1A-Gd1-N4-C16-46.5(4) N1-C1-C2-C3 −0.5(7) N1-Gd1-N4-C16 147.0(4) C22-C1-C2-C3−179.2(6) N3-Gd1-N4-C16 16.7(4) N1-C1-C2-C23 178.0(6) N1A-Gd1-N4-C16−69.1(4) C22-C1-C2-C23 −0.7(10) N2-Gd1-N5-C18 131.6(4) C1-C2-C3-C4−0.2(7) O5-Gd1-N5-C18 −125.9(4) C23-C2-C3-C4 −178.6(6) C1-C2-C3-C25178.7(6) C6-N2-C9-C10 −177.5(6) C23-C2-C3-C25 0.3(11) Gd1-N2-C9-C1014.2(8) C1-N1-C4-C5 180.0(6) C6-N2-C9-C8 −0.3(7) Gd1-N1-C4-C5 14.4(8)Gd1-N2-C9-C8 −168.6(4) C1-N1-C4-C3 −1.1(7) C7-C8-C9-N2 1.3(8)Gd1-N1-C4-C3 −166.7(4) C30-C8-C9-N2 −178.9(6) C2-C3-C4-N1 0.9(7)C7-C8-C9-C10 178.1(7) C25-C3-C4-N1 −178.1(5) C30-C8-C9-C10 −2.2(12)C2-C3-C4-C5 179.8(6) C11-N3-C10-C9 −179.7(6) C25-C3-C4-C5 0.9(10)Gd1-N3-C10-C9 −11.0(8) N1-C4-C5-C6 2.3(11) N2-C9-C10-N3 −-1.3(9)C3-C4-C5-C6 −176.5(6) C8-C9-C10-N3 −177.8(6) C9-N2-C6-C5 175.2(6)C10-N3-C11-C12 3.2(10) Gd1-N2-C6-C5 −18.7(9) Gd1-N3-C11-C12 −165.1(5)C9-N2-C6-C7 −0.8(7) C10-N3-C11-C16 −175.5(6) Gd1-N2-C6-C7 165.3(4)Gd1-N3-C11-C16 16.2(7) C4-C5-C6-N2 −1.5(11) N3-C11-C12-C13 −176.6(6)C4-C5-C6-C7 173.7(6) C16-C11-C12-C13 2.0(9) N2-C6-C7-C8 1.6(7)C31-O2-C13-C12 −2.3(9) C5-C6-C7-C8 −174.2(6) C31-O2-C13-C14 178.6(5)N2-C6-C7-C27 −175.9(6) C11-C12-C13-O2 177.4(6) C5-C6-C7-C27 8.3(10)C11-C12-C13-C14 −3.5(9) C6-C7-C8-C9 −1.7(7) C32-O3-C14-C15 −5.1(9)C27-C7-C8-C9 175.8(6) C32-O3-C14-C13 176.4(5) C6-C7-C8-C30 178.5(7)O2-C13-C14-O3 0.2(8) C27-C7-C8-C30 −4.0(11) C12-C13-C14-O3 −179.0(5)O2-C13-C14-C15 −178.3(5) C18-C19-C20-C21 3.0(7) C12-C13-C14-C15 2.4(9)C33-C19-C20-C21 179.5(6) O3-C14-C15-C16 −178.3(5) C18-C19-C20-C34−177.2(6) C13-C14-C15-C16 0.1(9) C33-C19-C20-C34 −0.7(11) C17-N4-C16-C15−6.6(9) C18-N5-C21-C22 −175.6(6) Gd1-N4-C16-C15 164.6(5) Gd1-N5-C21-C2216.5(9) C17-N4-C16-C11 173.3(6) C18-N5-C21-C20 1.8(7) Gd1-N4-C16-C11−15.6(7) Gd1-N5-C21-C20 −166.2(4) C14-C15-C16-N4 178.3(5) C19-C20-C21-N5−3.1(7) C14-C15-C16-C11 −1.6(9) C34-C20-C21-N5 177.1(6) N3-C11-C16-N4−0.6(8) C19-C20-C21-C22 174.1(6) C12-C11-C16-N4 −179.3(5)C34-C20-C21-C22 −5.7(10) N3-C11-C16-C15 179.3(5) N5-C21-C22-C1 3.9(11)C12-C11-C16-C15 0.6(9) C20-C21-C22-C1 −173.0(6) C16-N4-C17-C18 −179.0(5)N1-C1-C22-C21 −3.1(11) Gd1-N4-C17-C18 9.4(7) C2-C1-C22-C21 175.4(6)C21-N5-C18-C17 177.3(5) C3-C2-C23-C24 94.0(8) Gd1-N5-C18-C17 −12.4(7)C1-C2-C23-C24 −84.1(7) C21-N5-C18-C19 0.1(7) C2-C3-C25-C26 −96.7(8)Gd1-N5-C18-C19 170.5(4) C4-C3-C25-C26 82.0(8) N4-C17-C18-N5 1.4(9)C8-C7-C27-C28 −62.7(9) N4-C17-C18-C19 178.0(6) C6-C7-C27-C28 114.3(7)N5-C18-C19-C20 −2.0(7) C7-C27-C28-C29 −164.1(6) C17-C18-C19-C20−178.9(6) C27-C28-C29-O1 78.7(7) N5-C18-C19-C33 −178.6(6)C19-C20-C34-C35 90.3(8) C17-C18-C19-C33 4.6(11) C21-C20-C34-C35 −89.9(8)C20-C34-C35-C36 176.7(6) O5-Gd1-N1A-O1A −86.5(4) C34-C35-C36-O4 178.4(5)O1C-Gd1-N1A-O1A 85.8(5) N2-Gd1-O1A-N1A 172.0(4) O2A-Gd1-N1A-O1A−176.3(7) N5-Gd1-O1A-N1A −4.8(5) N1-Gd1-N1A-O1A −100.4(4) O5-Gd1-O1A-N1A83.2(4) N4-Gd1-N1A-O1A 115.4(4) O1C-Gd1-O1A-N1A −124.8(4) N3-Gd1-N1A-O1A50.8(4) O2A-Gd1-O1A-N1A 2.1(4) N2-Gd1-N1A-O2A 167.7(4) N1-Gd1-O1A-N1A105.3(4) N5-Gd1-N1A-O2A −7.5(4) N4-Gd1-O1A-N1A −61.5(4) O5-Gd1-N1A-O2A89.8(4) N3-Gd1-O1A-N1A −122.7(4) O1C-Gd1-N1A-O2A −97.9(5) N2-Gd1-O2A-N1A−16.7(5) O1A-Gd1-N1A-O2A 176.3(7) N5-Gd1-O2A-N1A 172.7(4) N1-Gd1-N1A-O2A75.8(5) O5-Gd1-O2A-N1A −80.2(4) N4-Gd1-N1A-O2A −68.3(4) O1C-Gd1-O2A-N1A117.2(4) N3-Gd1-N1A-O2A −132.9(4) O1A-Gd1-O2A-N1A −2.0(4) N2-Gd1-O1C-C1C−156.7(6) N1-Gd1-O2A-N1A −124.2(4) N5-Gd1-O1C-C1C −2.1(6) N4-Gd1-O2A-N1A104.0(4) O5-Gd1-O1C-C1C −95.5(6) N3-Gd1-O2A-N1A 46.8(4) O2A-Gd1-O1C-C1C54.3(7) Gd1-O1A-N1A-O3A 175.5(6) O1A-Gd1-O1C-C1C 135.8(6)Gd1-O1A-N1A-O2A −3.6(6) N1-Gd1-O1C-C1C −78.3(6) Gd1-O2A-N1A-O3A−175.5(6) N4-Gd1-O1C-C1C 67.2(6) Gd1-O2A-N1A-O1A 3.6(6) N3-Gd1-O1C-C1C133.8(6) N2-Gd1-N1A-O1A −8.6(4) N1A-Gd1-O1C-C1C 97.3(6) N5-Gd1-N1A-O1A176.3(4)

TABLE 13 Hydrogen bonds for 6 [Å and °]. D-H . . . A d(D-H) d(H . . . A)d(D . . . A) <(DHA) O1-H1 . . . O3B#1 0.84 2.04 2.851(7) 162 O4-H4 . . .O2B#2 0.84 2.08 2.887(7) 160 O1C-H1OC . . . O4#3 0.80 2.00 2.757(6) 157O5-H1WA . . . O1#4 0.81 1.97 2.758(6) 162 O5-H1WB . . . O3#1 0.81 2.102.878(6) 161

Symmetry transformations used to generate equivalent atoms:

#1 x,y+1,z #2 x,−y+½,z+½ #3 x,−y+½,z−½

#4−x+1,−y+1,−z+1

REFERENCES (SUPPLEMENTAL INFORMATION FOR EXAMPLE 3)

-   1. Sessler J L, Mody T D, Hemmi G W, Lynch, V M. Synthesis and    Structural Characterization of Lanthanide(III) Texaphyrins, Inorg.    Chem. 1993, 32: 3175-3187.-   2. Ehrlich, J.; Bogert, M. T., J. Org. Chem. 1947, 12: 522-534.-   3. Otwinowski Z, Minor W. Methods in Enzymology: Academic    Press; 1997. Altomare A, Burla M C, Camalli M, et al. SIR97: a new    tool for crystal structure determination and refinement. J. Appl.    Cryst 1999; 32:115-119.-   4. Sheldrick G M. SHELXL97. In: Program for the Refinement of    Crystal Structures. University of Göttingen, Germany; 1994. p.    Program for the Refinement of Crystal Structures.-   5. Wilson J C, editor. International Tables for X-ray    Crystallography Boston: Kluwer Academic Press; 1992.-   6. Sheldrick G M. SHELXTL/P C In. 5.03 ed. Madison, Wis.,    USA; 1994. p. Siemens Analytical X-ray Instruments, Inc.

Example 3—Development and In Vivo Quantitative Magnetic ResonanceImaging of Polymer Micelles Targeted to the Melanoma-Specific MarkerMC1R

Recent emphasis has been placed on the development ofrationally-designed polymer-based micelle carriers for theragnostics. Inthis study, micelles were decorated with a specific ligand for themelanocortin 1 receptor (MC1R), which has been evaluated as acell-surface marker for melanoma. The inventors describe the synthesisand characterization of a new gadolinium texaphyrin (Gd-Tx) chelate thatwas encapsulated in an IVECT™ micellar system, stabilized bycrosslinking with Fe(III) and targeted to MC1R. The inventors havedemonstrated that these stabilized Gd-Tx micelles are able to activelytarget MC1R expressing xenograft tumors in vitro and in vivo moreeffectively than control systems, including untargeted and/oruncrosslinked Gd-Tx micelles. Taken in concert, the findings reportedherein provide support for the conclusion that appropriately designedmicelles are able to deliver payloads to tumors expressing MC1R. Gd-Txis a known redox active agent; therefore, this approach may see eventualuse in the development of theragnostic agents.

Rationally-designed, polymer-based micelle carriers represent apromising approach to the delivery of therapeutic and/or diagnosticpayloads. They offer many potential advantages as delivery agents andcould serve to: (1) enhance the solubility of lipophilic drugs; (2)increase circulation times; and (3) lower the toxicity of the payload inquestion. Micelles with diameters between 20 and 200 nm are particularlyattractive since particles of this size can escape renal clearance. Thisgenerally translates into longer circulation times and can lead toimproved accumulation in tumor tissues as the result of an enhancedpermeability and retention (EPR) effect.^(1, 2) It has also beensuggested that selective accumulation in tumors relative to normaltissues can be enhanced through the use of tumor-specific cell-surfacetargeting groups, and that binding events may be used to trigger releasemechanisms. Such strategies are appealing since they could serve notonly to enhance uptake in tumor relative to normal tissues, but also toreduce the toxicity in peripheral organs.¹⁻³

Despite the advantages offered by micellar delivery systems, to date nomicellar system has been described that achieves the full promise oftargeting in vivo. Of additional concern is the fate of micelle deliverysystems in biological media.⁴ Though extensively studied in vitro, mostpublished delivery systems lack adequate information on micelleintegrity in vivo. Moreover, previously described micelle deliverysystems have suffered from an inherent instability in vivo, generallyundergoing collapse in the presence of serum lipids and proteins.⁴Micelles can be stabilized for in vivo use through crosslinking ofindividual acyl chains. To date numerous crosslinking reactions havebeen attempted, employing strategies that range from the use ofdisulfides^(5, 6) and other redox-sensitive bonds⁷ to temperature-⁸ andpH-sensitive functional groups.⁹⁻¹¹ Here, the inventors describe a novelcrosslinking procedure that relies on the pH sensitivity of metal-oxygencoordination bonds.^(12, 31) This particular form of crosslinking isknown to increase blood circulation times and result in a stable micelledelivery system that is able to selectively dissociate and release itscontents in the acidic tumor microenvironments.¹³

There are a number of micelle-based delivery systems for drugs such asdoxorubicin and paclitaxel currently in Phase I and Phase II clinicaltrials.^(1, 2) These systems do provide for increased circulation timesand larger area-under-the curve pharmacokinetics relative to thecorresponding free drug.² Some systems now in preclinical study are also“passively targeted,”^(6, 14, 15) meaning they lack any specific surfaceligands and rely solely on EPR to deliver their payload.^(5, 8, 16) Asignificant disadvantage with passive targeting of micelle deliverysystems is an increased probability for nonspecific delivery andaccumulation in clearance organs, such as liver and kidney, relative totumor.^(2, 17) Additionally, the significance of EPR in human cancersremains largely unproven and there is increasing evidence that EPR alonemay not be enough to ensure the selective delivery of a payload.¹⁷

Most attempts at micelle targeting have come from the use of ligandssuch as α_(v)β₃ (RGD), EGFR, or folate.^(7, 18-23) Unfortunately, mostof these targeted systems suffer from a high peripheraltoxicity,^(5, 7, 16, 19, 20) have only seen limited testing in vivo(e.g., in animal models lacking tumor xenografts^(21, 22)), or have notyet quantitatively demonstrated selective tumor accumulation relative toperipheral organs.^(7, 11, 18, 23, 24) It is also noteworthy, thatvarious other targeted systems have been reported to provide littleimprovement in tumor uptake as compared to their untargetedcontrols.^(7, 19, 20) Thus, there remains a need for more specificbiological targeting agents, including those that rely on localizationstrategies that are not EPR dependent. This may be of particularrelevance in clinical systems, where it has recently been proposed thathuman cancers have only a modest EPR, as compared to murinexenografts.¹⁷

One attractive target is the melanocortin 1 receptor (MC1R). Over 80% ofmalignant melanomas express high levels of this receptor.²⁵ Notsurprisingly, the MC1R has been investigated as a target for delivery ofimaging and therapeutic agents. Indeed, a number of MC1R ligands havebeen developed for this purpose.^(26-28, 29) The best known of these,[Nle⁴,DPhe⁷]-α-MSH (NDP-α-MSH),³⁰ is considered the “gold standard” forin vitro assays due to its ease of synthesis, low cost and high MC1Raffinity.^(29, 31) However, NDP-α-MSH is not selective for MC1R anddisplays strong nanomolar binding affinities to other melanocortinreceptor isoforms, e.g., MC4R and MC5R.³²⁻³⁴ Such off-target binding isundesirable given the presence of these receptors in the kidney, brainand CNS.³⁵⁻³⁹ Koikov et al. has reported the development of a ligand,4-phenylbutyryl-His-DPhe-Arg-Trp-NH₂ (SEQ ID NO:6), with highselectivity and specificity for MC1R²⁸. We have recently altered thisligand with an alkyne(4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂ (SEQ IDNO:3); 1)⁴⁰ for click attachment to a micelle-forming triblock polymer.Moreover, we have demonstrated in vitro that micelles decorated withcompound 1 retain the high binding affinity (2.9 nM K_(i)) of the freeligand and display improved target selectivity. In this prior work, theK_(i) of targeted crosslinked (XL) micelles for MC1R was found be tofour times lower than the corresponding targeted uncrosslinked (UXL)micelles while not binding to either of the undesired targets, MC4R orMC5R.⁴⁰ In this report we show how these micelles can be used to delivera contrast-enhancing agent to MC1R-expressing tumor xenografts.

Texaphyrins are a series of expanded porphyrins that have attractedinterest in the area of cancer research.^(37, 41-44) Gadoliniumcomplexes of texaphyrin (Gd-Tx) have been specifically evaluated innumerous clinical trials, including those for metastatic cancer to thebrain, non-small cell lung cancer (NSCLC) and non-Hodgkin's lymphoma.⁴³Due to its high electron-affinity⁴³, Gd-Tx is able to capture electronsfrom reducing metabolites and transfer them to endogenous oxygen,thereby triggering cell death via oxidative stress without modificationof cellular DNA.^(41, 44) Texaphyrins have been tested as radiationsensitizers in both animal and human trials.⁴⁰⁻⁴³ Additionally, theincorporation of gadolinium into the texaphyrin macrocycle allows thetissue distribution of Gd-Tx to be studied non-invasively via standardmagnetic resonance imaging (MRI) methods.^(42, 44) The combined imagingand therapeutic features of Gd-Tx makes it attractive as a theragnosticagent. One way to realize this promise would be to enhance tumorlocalization through use of a targeted micelle. Here, we show that thiscan indeed be achieved through use of crosslinked systems conjugated to1.

To develop micelles containing Gd-Tx, the inventors have taken advantageof a triblock polymer micelle system with enhanced stability (IVECT™)that was initially developed by Intezyne Technologies Inc. (Tampa,Fla.).^(13, 40) This triblock polymer is composed of a hydrophobicencapsulation block, a responsive stabilizer block, and a hydrophilicmasking block that contains an azide for functionalization via clickchemistry. The main advantage of IVECT™ micelles over traditionalmicelles is the incorporation of the stabilization block, which allowsthe micelles to be crosslinked via a pH-sensitive Fe(III) metalcoordination reaction.^(12, 13, 40) They are also biodegradable anddesigned to release their payload in the acidic microenvironment oftumors.¹³ As detailed below, this approach has allowed for thegeneration of a stabilized IVECT™ micelle system that incorporates Gd-Txand which both penetrates into xenografted tumors with high selectivelyand clears from circulation without being retained in the kidney orliver. Tumor penetration, as inferred from MRI studies, was not observedwith either untargeted or uncrosslinked micelles. On this basis, theinventors propose that the present approach provides for tumor-specifictargeting that is superior to that provided by EPR alone.

Results

A. Gadolinium-Texaphyrin (Gd-Tx) Structure

The Gd-Tx complex used in this study was prepared using a syntheticprotocol similar to those published previously⁴⁵⁻⁴⁷ (cf. FIG. 39). Asingle crystal X-ray diffraction analysis of the Gd-Tx complex confirmedthe expected planar structure for the core macrocycle and revealedseveral ancillary ligand and solvent interactions (FIG. 34).

B. Physical Properties of the Gd-Tx Micelles

The targeted Gd-Tx micelles were prepared using a novel optimizedencapsulation strategy (FIG. 34). The average particle size wasdetermined using standard dynamic light scattering (DLS) methods. Thesurface charge and gadolinium percent loading by weight were determinedby zeta potential and elemental analyses, respectively (Table 6). Thesestudies provided support for the notion that there are no differences inmicelle size for the crosslinked (XL) and uncrosslinked (UXL) pairs, orfor the targeted (T) and untargeted (UT) pairs. However, significantdifferences in micelle size were seen depending on the extent ofGd-weight loading. For example, micelles with 5% Gd-Tx (wt:wt) weresignificantly larger (169±40 nm) than those containing 0.5% (87±40 nm)(p≤0.01; vs. 5%) or 0.05% (84±24 nm) Gd-Tx (p≤0.01; vs. 5%). However,the size of the latter two micelle systems, both of which arecharacterized by relatively low Gd-Tx loading levels, were notsignificantly different. Attempts to create formulations with Gd-Txweight loading percentages above 10% proved unsuccessful. Particlecharges ranged from −0.33 to −29 mV as deduced from zeta potentialanalyses.

TABLE 6 % Gd-Tx % Gd-Tx DLS Sample Encap. Encap. Charge Size #Stability* Targeting** (calculated) (actual) (mV) (nm) 1 UXL UT 5.4 5.3−26.61 208.20 2 UXL T 5.4 5.2 −29.23 113.60 3 XL UT 5.3 5.1 −11.12174.70 4 XL T 5.2 5.0 −13.73 179.40 5 UXL UT 0.54 0.51 −17.70 88.90 6UXL T 0.53 0.50 −17.74 88.80 7 XL UT 0.52 0.52 −10.73 87.50 8 XL T 0.510.51 −9.49 82.50 9 UXL UT 0.054 0.049 −20.33 104.80 10 UXL T 0.053 0.05−17.28 49.40 11 XL UT 0.052 0.052 −2.62 87.30 12 XL T 0.051 0.051 −0.3393.50 *Micelles are stabilized with Fe(III) crosslinking (XL) reaction.UXL denotes uncrosslinked micelles. **Micelles are targeted (T) with anMC1R-specific ligand. UT denotes untargeted micelles.c. Gd-Tx Micelle Stability

Uncrosslinked Gd-Tx (20 mg/mL in PBS) micelles at 5% weight loading weredialyzed for 6 hours against PBS (pH 7.4). Elemental analyses (ICP-OES,Galbraith Labs) of the Gd-Tx micelles pre- and post-dialysis indicatedthat the uncrosslinked micelles retained 60% of the encapsulated Gd-Txafter dialysis.

D. Competitive Binding Assays

Time-resolved lanthanide fluorescence competitive bindingassays^(33, 40) were performed in an effort to optimize ligand loadingfor maximal avidity. In these assays, increasing concentrations ofmicelles were measured for their ability to competitively displaceEu-labeled NDP-α-MSH 2. The remaining Eu was then measured using timeresolved fluorescence (TRF, see Methods). As gadolinium(III) cations canpotentially interfere with the lanthanide-based TRF binding assays,³³unloaded triblock polymer micelles (i.e., free of Gd-Tx) targeted with2.5% to 30% ligand 2 by weight loading (see FIG. 34) were used. Micellesstabilized with Fe(III) crosslinking had the highest binding avidity at5% ligand loading, as reflected in the lowest K_(i) (1.49±0.12 nM K_(i),n=4). It was also observed that XL micelles had significantly higherbinding avidities at all ligand loading levels (p<0.001). In vitroassays were also conducted with ligand 1-targeted XL and UXL micelles at10% ligand loading, as well as 1-targeted monomers (see FIG. 34).⁴⁰ TheK_(i) of the 1-targeted XL micelles (2.9±0.42 nM; n=4) was 4 times lowerthan the corresponding UXL micelles (12±2.6 nM; n=4).⁴⁰ Control assayswith untargeted micelles (XL and UXL) and untargeted polymer revealed nodetectable interaction with the receptor.

E. In Vitro MR Imaging

To determine the MRI relaxivity of Gd-Tx labeled micelles, phantoms wereconstructed in which each sample (T-XL, T-UXL, UT-XL, UT-UXL micelles)was prepared at the same Gd-Tx concentration (0.01 mg/mL) (FIG. 36). T₁values were determined by progressive saturation relaxation measurementsusing an Agilent 7T small animal MRI spectrometer using a spin echosequence, SEMS (see Methods) and T₁ values for each cohort of sampleswere averaged. There was no apparent T₁ effect attributable to thedifferent micelle formulations, with coefficients of variation (CVs)ranging from 0.02 to 0.1 for each row of T₁ measurements. Individual T₁values for each well are provided in the Supplemental Information(Supplemental FIG. 44). The phantom studies served to confirm theexpectation that the observed T₁ values are positively correlated withGd-Tx weight loading, with 0.05% encapsulated Gd-Tx providing the lowestmean T₁ value (1.6 s).

F. In Vivo MR Imaging

SCID mice with subcutaneous MC1R-expressing tumors were injected with0.5% and 5% w/w Gd-Tx micelles (T-XL, T-UXL, UT-XL, UT-UXL) via tailvein at a dose of 12 μmol Gd-Tx/kg. Targeted micelles (T-UXL and T-XL)were formulated with 5% (w/w) of 2-targeted polymer. Using an Agilent 7Tsmall animal MRI spectrometer, coronal T_(i)-weighted spin echo multislice (SEMS) images were acquired of each animal prior to and 1, 4, 12,24 and 48 h after injection of the micelles. Following imaging, MC1Rexpression was confirmed in each tumor by immunohistochemistry staining(FIGS. 43A-B). FIG. 37A shows representative images of the center slicesof the tumors of animals injected with the different 0.5% Gd-Tx loadedmicelles recorded at different time-points. FIG. 37B shows thepre-injection and 24 h post-injection image of the T-XL formulationloaded with 5% Gd-Tx (w/w).

To quantify enhancement due to tumor uptake of the micelles, intensityhistograms for right (R) and left (L) whole tumors, kidneys and liverswere prepared using a MATLAB program (Mathworks, Natick, Mass.) bydrawing a region of interest (ROI) across all applicable slices for eachtime point. A mean intensity value was then calculated and normalized tothigh muscle (see Methods Section and Supporting Information for moredetails). FIGS. 38A-D show the tumor uptake and clearance data for each0.5% Gd-Tx micelle group in tumor (FIG. 38A), kidneys (FIG. 38C) andliver (FIG. 38D). The T-XL micelle group is the only one to showsignificant contrast enhancement in the tumors (4b), with a peakaccumulation occurring at 24 h (FIG. 48D). The 5% Gd-Tx loaded micellesdid not show significant tumor enhancement at any time-point for anytargeting or crosslinking formulation (FIG. 37B). The increasedenhancement in the tumors of animals injected with the 0.5% Gd-Tx T-XLmicelles can be visualized in the post-injection MR images (FIG. 37A,top row) relative to tumors in all other animals injected with thecontrol formulation (UT-XL, T-UXL, UT-UXL). Again, no other micellegroup displayed visible tumor uptake. The contrast enhancement for theT-XL micelles peaked in the kidneys at 4 h and steadily decreasedthereafter, whereas enhancement in the liver peaked at 1 h.

To test whether the enhancement observed in the 0.5% Gd-Tx, T-XL tumorswas statistically different from the other groups, a 1-way ANOVAanalysis (Dunnett's Multiple Comparison Test) was carried out. While nosignificant difference was observed among the groups at 1 h, the T-XLgroup was statistically different from all other groups at 4 h to 48 h(p<0.001 for 4-24 h; p<0.05 at 48 h). Additional analyses using theStudent's t-test revealed that there are no statistical differencesamong the control groups (UT-XL, T-UXL or UT-UXL) at any time point.

In vitro europium time-resolved fluorescence competition binding assaysconducted with both 1- and 2-targeted micelles provide support for thecentral hypothesis underlying this study, namely that crosslinkingprovides stability to the micelle system and that the composition of themicelle can be modified to allow for targeting. NDP-α-MSH 2 was chosenas a model ligand for competition due to its relatively high affinityfor MC1R (1.9 nM), and for the ease of synthesis that itprovides.^(29, 31) In the percent targeting optimization assays with analkyne-functionalized NDP-α-MSH (2, FIG. 35), there was a cleardifference between the binding affinities of the crosslinked (XL) anduncrosslinked (UXL) micelles. This finding is ascribed to the Fe(III)crosslinking, which serves to stabilize the micelles in biologicalmedia. In the absence of crosslinking, the micelles dissociate, in wholeor in part, to free monomers, leading to a loss of structural integrityand the premature release of the payload (the encapsulated contrastagent in the present instance). A second advantage of crosslinking isthat it leads to an operational increase in binding avidity, a resultthat may reflect a benefit of multivalent interactions. The 1-targeted(T) micelles of this study also exhibited a stronger avidity to the MC1Rreceptor when crosslinked (T-XL) as compared to their uncrosslinkedcounterpart (T-UXL), a finding we take as further support for thecontention that i) crosslinking stabilizes micelles and ii) multipleligands on the micelle surface provide for enhanced binding.

In vitro phantom experiments demonstrated that micelles with a lowerpercentage of Gd-Tx loading (w/w) produced shorter T1 values (FIG. 36).Moreover, in vivo experiments served to confirm that those with 5% Gd-Txloading (w/w) provided no measurable contrast enhancement (relative tobackground) in tumor xenografts. At first blush, these observationsappear counter-intuitive since higher contrast enhancement might beexpected at higher gadolinium concentrations. However, gadoliniumoverloading, wherein encapsulated gadolinium is less accessible to waterat higher concentrations due to “overcrowding” within the micelle, wouldgive rise to a lower level of enhancement. The lack of differentiationin the T₁ values of the crosslinked and uncrosslinked micelles at lowerloading levels is thought to result from overcrowding. However,overcrowding does not provide an explanation for the lack ofdifferentiation between the crosslinked and uncrosslinked micelles atequal concentrations. We believe this result is best explained by adifferent effect, namely localization of the Gd-Tx complex within thestabilized (hydrophilic) block of the micelle, rather than within thecore. This portion of the polymer is composed of poly(aspartic acid), towhich Gd-Tx is known to coordinate,⁴⁸ and is expected to mask thedifferences is micelle stabilization.

Based on the results of the phantom experiment and the lack ofsignificant in vivo tumor contrast enhancement in the case of the 5%(w/w) Gd-Tx micelles, the micelles with 0.5% Gd-Tx (w/w) micelle loadingwere chosen for the in vivo experiments. In accord with the designexpectations, these in vivo experiments revealed improved MRI contrastenhancements upon administration of the Gd-Tx containing T-XL micelles,with maximal enhancement observed at 24 h. As can be seen by aninspection of FIGS. 37A-B and 38A-D, this enhancement was not seen withthe other micelle systems, supporting the contention that the T-XLmicelles provide good systems for effecting tumor localization andimaging.

The unique ability of the T-XL micelles to penetrate the tumor appearsto result from a combination of the MC1R-specific targeting group andthe enhanced stability provided by the Fe(III) crosslinking. Iftargeting alone were enough to produce effective tumor enhancement, wewould also observe a substantial uptake in the case of the T-UXLmicelles. Likewise, if crosslinking and EPR alone were enough to affectaccumulation, we would observe an increased build-up in the UT-XL group.Finally, it is important to note that the enhancement observed in theT-XL group was not the result of free Gd-Tx (which is known toaccumulate in tumors selectively^(42, 43, 49, 50)). If this were thecase, we would have observed enhanced uptake in all four micelle groups(i.e., UT-UXL, T-UXL and UT-UXL, in addition to the T-XL system). Thiswas not seen. Thus, the in vivo data are consistent with the conclusionthat the Gd-Tx containing T-XL micelles allow for functionallyacceptable binding avidity, stability, tumor penetration and uptake.Presumably, the crosslinking reaction stabilizes the micelles afteradministration and during initial time points while they circulatethroughout the bloodstream, while the targeting group allows the systemto bind to, and be retained within, the tumor cells.

The present inventors have reported the development of a ligand specificto MC1R and have shown that the conjugation of this ligand to the IVECT™micelle system does not result in a significant decrease in bindingavidity.⁴⁰ In this Example, the inventors describe the synthesis,incorporation and characterization of a new gadolinium texaphyrin(Gd-Tx) that is characterized by a high inherent T₁ relaxivity. Theinventors also detail its encapsulation within the IVECT™ system and theproduction of crosslinked micelles by reaction with Fe(III). Moreover,we have demonstrated that the targeted Gd-Tx micelles are selectivelyretained in target-expressing xenograft tumors in vivo. To the best ofour knowledge, this is the first example of a targeted micelle that iscapable of carrying a payload and which outperforms systems based on EPRin terms of tumor penetration, uptake and retention.

Advantages of the current system include the following: (1) the target,MC1R, is highly expressed in melanoma cells and not in healthy tissues,except for melanocytes; (2) high short term stability, and (3) anability to accumulate in tumors, rather than various clearance organs.These attributes are reflected in the in vivo images that reveal uptakedeep within the tumor with peak accumulation at 24 h. In contrast, peakkidney and liver accumulations were seen at 1-4 h. These differences arethought to reflect the benefits of targeting. However, biodegradation ofthe stabilized micelles may contribute to the effect; to the extent itoccurs on short time scales (on the order of hours), it would allow forrelease of payload (Gd-Tx) within the tumor while concurrently clearingfrom circulation. While further investigations will be required todetail the full pharmokinetic profile of these new micelles, it isimportant to appreciate that from an operational perspective theyconstitute the first examples of systems that are capable of deliveringa payload in a tumor selective fashion.

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We claim:
 1. A method of treating melanoma in a human or non-humananimal subject, comprising administering an MC1R-targeted agent to thesubject, wherein the MC1R-targeted agent comprises a MC1R peptide ligandand a moiety, wherein the moiety comprises an anti-cancer agent, andwherein the MC1R peptide ligand and the moiety are covalently linked bya reaction product of a first functionality coupled to the MC1R peptideligand and a complementary second functionality coupled to the moiety,wherein the MC1R peptide ligand comprises: (SEQ ID NO: 3)4-phenylbutyryl-His-DPhe-Arg-Trp-Gly-Lys (hex-5-ynoyl)-NH₂; or(SEQ ID NO: 4) H-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂; or  (SEQ ID NO: 5)H-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)- Arg-DPhe-Asp-Arg-Phe-Gly-NH₂;  or (SEQ ID NO: 6)4-phenylbutyryl-His-DPhe-Arg-Trp,

and a functional group coupled to the C-terminus of the MC1R peptideligand.
 2. The method of claim 1, wherein the MC1R peptide ligandcomprises 4-phenylbutyryl-Hi s-DPhe-Arg-Trp-Gly-Lys(hex-5-ynoyl)-NH₂(SEQ ID NO:3).
 3. The method of claim 1, wherein the MC1R peptide ligandcomprisesH-Lys(hex-5-ynoyl)-Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂(SEQ ID NO:4).
 4. The method of claim 1, wherein the MC1R peptide ligandcomprisesH-Lys(hex-5-ynoyl)Tyr-Val-Nle-Gly-His-DNal(2′)-Arg-DPhe-Asp-Arg-Phe-Gly-NH₂(SEQ ID NO:5).
 5. The method of claim 1, wherein the MC1R peptide ligandcomprises 4-phenylbutyryl-His-DPhe-Arg-Trp (SEQ ID NO:6).
 6. The methodof claim 1, wherein the subject is human.
 7. The method of claim 1,wherein the subject is a non-human animal.
 8. The method of claim 1,wherein the melanoma is metastatic melanoma.
 9. The method of claim 1,wherein the anti-cancer agent kills melanoma cells or inhibits growth ofmelanoma cells.
 10. The method of claim 1, wherein the anti-cancer agentis a chemotherapeutic agent.
 11. The method of claim 1, wherein theanti-cancer agent is a cytotoxic agent.
 12. The method of claim 1,wherein the anti-cancer agent is a taxane.
 13. The method of claim 1,wherein the anti-cancer agent is one or more selected from amongmelphalan, ifosfamide, dacarbazine, paclitaxel, and vincristine.
 14. Themethod of claim 1, wherein the MC1R-targeted agent is administeredsystemically.
 15. The method of claim 1, wherein the MC1R-targeted agentis administered locally at a site of the melanoma.
 16. The method ofclaim 1, wherein the MC1R-targeted agent is administered orally orintravenously.
 17. The method of claim 1, wherein the MC1R-targetedagent is in a pharmaceutical composition comprising the MC1R-targetedagent and a pharmaceutically acceptable carrier, and wherein thepharmaceutical composition is administered to the subject.
 18. Themethod of claim 17, wherein the pharmaceutical composition isadministered by intratumoral injection.